Chi osan-based nanomedicine o osma inic acid ocula deli e y
Thesis p esen ed o ob ain he PhD deg ee in Pha maceu ical Sciences,
Pha maceu ical Technology Special y,
Facul y o Pha macy o Uni e si y o Po o
by
Sa a Isabel Macedo Bap is a da Sil a
Unde supe ision o P o . D . B uno Sa men o and co-supe ision o P o . D . Domingos
Fe ei a and P o . D . Manuela Pin ado
Oc obe , 2014
i
Decla a ion
The pa ial ep oduc ion o his hesis is au ho ized only o esea ch pu poses by w i en
decla a ion o he pe son conce ned.
(Sa a Bap is a da Sil a)
Inspi a ion
“Success is a jou ney, no a des ina ion!”
Ben Swee land

i
Dedica ion
“The e a e only wo ways o li e li e.
One is as hough no hing is a mi acle.
The o he is as hough e e y hing is a mi acle.”
Albe Eins ein
I dedica e his hesis o he mos impo an pe sons in my li e,
my amily, Osca and ou daugh e ,
Ca a ina Isabel.
.
ii
Acknowledgemen s
“Lo d I can’ say i in wo ds…can you please jus lis en h ough my hea ”
Unknown
I would like o o mally exp ess my deep g a i ude o he ollowing people and ins i u ions –
who (and which) ha e mean a lo o me du ing my PhD p og am, and made i possible:
Fundação pa a a Ciência e a Tecnologia, o inancial suppo ia a PhD ellowship ( e .:
SFRH/BD/61423/2009), unde he supe ision o P o esso B uno Sa men o; said g an
pe mi ed imely de elopmen o my esea ch p og am, as well as pa icipa ion in se e al
in e na ional scien i ic mee ings o complemen my aining and sha ing my esul s.
Labo a o y o Pha maceu ical Technology, Facul y o Pha macy, Uni e si y o Po o o
accep ing me as a PhD s uden , o he hospi ali y and wo k condi ions a ailable, du ing
my doc o al p og am.
My mos since ely acknowledges o Escola Supe io de Bio ecnologia o Uni e sidade
Ca ólica Po uguesa (ESB-UCP), o he c ucial collabo a ion in my PhD cou se, he
indubi able hospi ali y and o p o iding acili ies and logis ical o bes suppo my s udies.
INEB – Ins i u o de Engenha ia Biomédica, ISCS–N – Ins i u o Supe io de Ciências da
Saúde – No e and IBILI-Ins i u e o Biomedical Imaging and Li e Sciences – Uni e si y o
Coimb a, o he accep ance, kindness and cons an a ailabili y coope a ion du ing his
p ojec .
P o esso B uno Sa men o my supe iso , who I am since ely g a e ul o ha ing accep ed
me as a PhD s uden , o he wide suppo and comp ehensi e scien i ic guidance he
con inuously ga e me. His consis en and in eg a ed con ibu ion o my g ow h, bo h as a
pe son and as a esea che , has been by all means ou s anding. I would like, in pa icula ,
o hank him o e e y e o made in gua an ee he bes esea ch condi ions, going
whe e e necessa y o ind he mos app op ia e suppo ; and o his e e las ing
encou agemen , pa ience and mo i a ion, as well as his a ailabili y o discuss speci ic and
gene al opics o my disse a ion. I am indeed deeply g a e ul, o ha ing always belie ed
in my abili ies, o all he conce ns, p oblems, oppo uni ies and achie emen s sha ed
along his jou ney, o he iendship and a ec ion, my hea el hanks.
iii
P o esso Manuela Pin ado my co-supe iso , I would like o hank o always accep ing
me as a s uden , ha ing accompanied du ing hese 12 yea s o academic aining, o
always belie ing in my abili ies, skills and compe ences, o he uncondi ional
unde s anding, pa ience, iendship and complici y. Fo he belie ed and dedica ion o my
wo k, o all he con e sa ions es ablished and knowledge sha ed h oughou hese yea s
and du ing his doc o al cou se, as well as o he encou agemen , a ec ion and help; and
also o p o iding a heal hy, happy and ex emely p o essional wo king en i onmen , my
deepes hanks.
P o esso Domingos Fe ei a my co-supe iso , I would like o hank o his sympa hy,
kindness, cons an a ailabili y and willingness o help du ing my doc o al p og am. Fo
e e y e o o ensu e he bes ins i u ional ecep ion condi ions and e e y logis ical ha
could ensu e he success o my wo k, my since e g a e ul.
P o esso F ancisco Amb ósio, my deepes g a i ude o ag eeing o collabo a e in his
PhD p ojec . Fo all he help, coope a ion, unde s anding, by all he e o s made he
in es men o ime and esou ces in be e moni o ing and pe o mance o his wo k. Fo
ha ing gi en me he oppo uni y o wo k wi h his esea ch eam and by he unma ched
amiabili y.
My mos since e hanks o P o esso Ho acina Ca alcan e and o he en i e S am o d
amily: P o esso New on, P o esso Tania, Thayza and Tha hiana, o ha e accompanied
me du ing my doc o al plan in e e y possible way, o all he help, dedica ion, and
eno mous iendship.
My colegues and iends wi hin he di e en esea ch g oups – José das Ne es, Fe nanda
And ade, Filipa An unes, Ped o Fon e, F ancisca A aújo, Ru e Nunes, Ca la Pe ei a,
Manuela Amo im, Ana Oli ei a, Raquel Madu ei a, Débo a Campos, Raquel Boia, Filipe
El as, Tiago Ma ins, Ped o T alhão, Ma ia Madei a, Joana Ma ins, and so many o he s.
By somehow helping me in de eloping my s udies and sha ed by so many di icul ies and
achie emen s, which ce ainly has helped me g ow as a pe son and as a p o essional.
My closes iends which a e he amily ha I chose o me e e y day:
Helena Mon ei o, hank you o you iendship which al eady makes “sil e wedding”, o
ou pe pe ual oa h on ime, o always ind you sel wi hou seeking, o e en a in
dis ance seems like I ha e been wi h you yes e day, and always.
ix
Sand a Bo ges, wo ds will be o su e all sho in exp essing my e e nal iendship. Thank
you o b inging a ain-bow o my li e, o he c aziness sha ed. Fo e e y conce n, wo y,
and happiness momen s join , o lis ening, ad ising, o he cons an pa ience and
complici y; Fo being always he e.
Manuela Amo im, I will no be able o hank you e e y hing ha you a o me and
e e y hing wha you al eady has done o me du ing his p ojec , much less in a nu shell.
Abo e all hank you o he uncondi ional iendship, complici y, and con idence, o e e y
laugh and ea sha ed, o being my ue iend.
Inês C a o Roxo, Joana Ba bosa, F anklin Cos a (and he li le Ma ilde), Rica do F eixo,
Luciana Sil a and Rena o Resende hank you o always accompanied my li e a i s bes
and wo s o e e y momen spen , I app ecia e and ecip oca e wi h e e nal iendship.
I also ha e o lea e my deepes g a i ude o my swee F ancisca Ma ia, who accompanied
my li e o e e , and hank he o he immense dedica ion o my amily, o hei undying
a ec ion and uncondi ional suppo in e e y momen o ou li e, om bes o wo s ; My
mos since e and p o ound hanks.
I would like o hank o my b o he Jo ge Filipe and my sis e So ia Manuel, being my bes
iends, o exis ing and making my li e so ull ield, so comple e; Fo being wi h me in
e e y d eam, igh , s ep and o ne e li e me alone. I would also ha e o hank o my
nephews (Ca los Edua do, Filipa Alexand a and Ba ba a So ia), o being he bes
con inui y o my b o he s, o hei lo e and o being always wi h me in a heal h madness.
My G andmo he , Isabel Ma ia, will always be my sunshine, my companion o all hou s,
he example o s eng h and ligh , and he way o being and li ing will always inspi e my
li e.
Wo ds will be educ i e and insu icien o hank my pa en s o he uncondi ional lo e, o
making me wha I am oday, o e e y hing hey ha e gi en me h oughou my li e, by
making he de elopmen o my academic aining possible and o always belie ed in me.
Thank you o he uncondi ional lo e, s eng h, suppo and cou age injec ed in me, oday
and e e y day. Thank you o e e y hing, now and o e e .
And las , bu a om leas – Osca , is my Al e Ego, my companion o d eams and
s uggles. Osca is he be e hal o me, he mo e awa e, esponsible and ealis ic. I g ew
up wi h him pe sonal and in ellec ually. He has accompanied e e y momen o my li e,
x i
96,1 ± 0,2 e 98,2 ± 0,1% pa a as nanopa ículas de ácido osma ínico, sal a e segu elha,
espe i amen e. Es es alo es mais ele ados associados à nanoencapsulação dos
ex a os ambém podem se associados à meno quan idade de ácido osma ínico nas
nanopa ículas de qui osano, uma ez que a concen ação inicial é documen ada como
in e samen e p opo cional à p óp ia e iciência de associação. O pe il de libe ação in i o
do ácido osma ínico oi a aliado em ampão os a o (PBS), a pH 7,4 nas di e en es
o mulações, po um pe íodo de 60 min, e não se obse a am di e enças signi ica i as (P
> 0,05). A ápida libe ação do ácido osma ínico dá indicações que es es sis emas de
nanopa ículas podem o nece uma es a égia acional pa a o desen ol imen o de
o mulações de libe ação imedia a pa a adminis ação ocula do ácido osma ínico. A
e iciência de associação e de libe ação in i o o am ealizadas u ilizando um mé odo de
c oma og a ia líquida de al a e iciência (HPLC), especialmen e desen ol ido e o imizado
pa a ga an i a ob enção de esul ados p ecisos e exa os. As análises de calo ime ia
di e encial de a imen o (DSC) e a espe o o ome ia de in a e melho po ans o mada
de Fou ie (FTIR) pe mi i am conclui que não o am encon adas in e ações químicas
en e os an ioxidan es e o qui osano, depois do p ocesso de encapsulação. A a i idade
an ioxidan e dos nanosis emas oi a aliada pelos mé odos de 2,2-azinobis-(3-e il-
benzo iazolin-6-ácido sul ónico) (ABTS) e de capacidade de abso ção adical (ORAC),
an es e depois do p ocesso de lio ilização, pa a ga an i que a a i idade an ioxidan e não
é comp ome ida du an e o p ocesso de secagem das pa ículas. Os melho es esul ados
de a i idade an ioxidan e o am ob idos pelo mé odo de ORAC após lio ilização das
pa ículas, os esul ados pa a as nanopa iculas de ácido osma inico, sal a e segu elha
o am: 3,6520 ± 0,1770, 0,4251 ± 0,0069 e 0,4526 ± 0,0087 µmol/eq T olox,
espe i amen e. Toda ia oi obse ada uma a i idade an ioxidan e mais baixa nas
nanopa ículas do que nos compos os li es, de ido ao e ei o da nanoencapsulação. As
pa ículas demons a am p op iedades mucoadesi as após incubação com mucina, pelo
aumen o em amanho e consequen e diminuição da ca ga de supe ície. Os esul ados
indicam que pode se expec á el um aumen o do empo de e enção sob e a mucosa
ocula após a ins ilação. Todas as o mulações demons a am se segu as pa a o es e
de ci o oxicidade 3-(4,5-dime il iazol-2yl)-2,5-di enil b ome o de e azolina (MTT) e pa a o
es e da libe ação da enzima lac a o desid ogenase (LDH), sem ci o oxicidade ele an e
(abaixo de 10%, pa a odas as o mulações e concen ações), em linhas ocula es da
e ina (epi élio pigmen a da e ina - ARPE-19) e da có nea (linha de células da có nea
humana - HCE-T). O es e da memb ana co ioalan óide (HET-CAM Tes e) oi u ilizado
como al e na i a aos es es biológicos em coelhos ( es e de D aize) e ambém suge e a

x ii
ausência de i i ação das pa ículas no olho. Os es udos de pe meabilidade em
monocamada de células da có nea (HCE) e elou um coe icien e de pe meabilidade
apa en e (Papp) de 3,41 ± 0,99 x 10-5 e 3,24 ± 0,79 x 10-5 cm / s pa a as nanopa ículas de
ácido osma ínico e pa a o ácido osma ínico li e, espec i amen e. O es udo de
pe meabilidade em monocamada de células da e ina (ARPE-19) e elou alo es de Papp
de 3,39 ± 0,18 x 10-5 e 3,60 ± 0,05 x 10-5 cm/s pa a as nanopa ículas de ácido
osma ínico e pa a o ácido osma ínico li e, espec i amen e. Não hou e di e ença
signi ica i as (P > 0,05) en e os alo es de pe meabilidade das nanopa iculas, compos o
li e e en e ambas as linhas celula es, p o a elmen e de ido ao pe il de libe ação
ápido das nanopa ículas acima desc i o. Fo am ei os es es p elimina es in i o, em
que o ácido osma ínico oi inje ado na ca idade in a í ea de a os Wis a , num modelo
animal isquemia- epe usão (I-R). Ele o e inog amas (ERG) e ensaios imuno-
his oquímicos e ela am que o ácido osma ínico (a uma concen ação de: 50 µM), po
injeção in a í ea, não e e um e ei o p o ec o na e ina. O que pode á se de ido danos
p ó-in lama ó ias se e os no modelo I-R, di íceis de e e e com o es udo de uma única
injeção. No en an o, e conside ando os bons esul ados ob idos nes e abalho, as
pa ículas de qui osano con endo ácido osma ínico demons a am se segu as,
mucoadesi as, com ele ado po encial de pe meabilidade ocula e com um bom pe il de
a i idade an ioxidan e, o que pe mi e conclui que es es nanosis emas podem se
impo an es pa a a p e enção de doenças degene a i as ocula es. Os esul ados des a
ese, pe mi em ambém conclui que es es nanosis emas na u ais são p omisso es na
adminis ação ópica de an ioxidan es no olho e essal a a necessidade de se explo a
no os sis emas pa a ul apassa as limi ações na e iciência da adminis ação ópica de
á macos no olho.
Pala as-cha e: Qui osano, ácido osma inico, ex a os, doenças ocula es
x iii
This wo k was submi ed as a PhD Thesis in pa ial ul ilmen o he equi emen s o
Philosophiæ Doc o (PhD) deg ee in Pha maceu ical Sciences a he Facul y o
Pha macy, Uni e si y o Po o.
I was conduc ed unde he guidance o P o . D . B uno Filipe Ca melino Ca doso
Sa men o, PhD, A ilia ed Resea che a INEB - Ins i u o de Engenha ia Biomédica and
Assis an P o esso a Ins i u o Supe io de Ciências da Saúde-No e (ISCS-N), and unde
he co-supe ision o P o . D . Ma ia Manuela Es e ez Pin ado, Assis an P o esso a
Bio echnology School o Po uguese Ca holic Uni e si y and P o . D . Domingos Ca alho
Fe ei a, Full P o esso o Facul y o Pha macy, Uni e si y o Po o.
The esea ch expe imen al wo k was conduc ed a he Labo a o y o Pha maceu ical
Technology, Facul y o Pha macy, Uni e si y o Po o, in collabo a ion wi h CBQF -
Bio echnology School o Po uguese Ca holic Uni e si y, INEB - Ins i u o de Engenha ia
Biomédica, ISCS–N - Ins i u o Supe io de Ciências da Saúde – No e and IBILI - Ins i u e
o Biomedical Imaging and Li e Sciences – Uni e si y o Coimb a.
xix
Scope and ou line
This hesis was o ganized in 9 chap e s, hus closely e lec ing he de elopmen o my
esea ch wo k. All chap e s we e ela ed o each o he and he aims and me hodology
chosen in each chap e we e indeed dependen on he conclusions b ough abou in
p e ious one(s).
O e all, he wo k desc ibed in his hesis encompasses de elopmen and cha ac e iza ion
o chi osan nanopa icles o he osma inic acid, sage and sa o y encapsula ion - o
p e en and con ol degene a i e eye diseases.
Pa I include Chap e 1, and en ail a bibliog aphic e iew ega ding chi osan biological
p op ie ies, biomedical po en ial as well as chi osan-based deli e y sys ems. A pa icula
emphasis was pu on he key ac o o an ioxidan s in he degene a i e eye diseases
p ophylaxis, as well as in he nanoca ie s as a way o imp o e an ioxidan ac i i y
pe o mace and e icacy. In Pa II - Chap e 2, he p ojec aims and goals we e de ailed
o be a guideline o he wo k majo co e. In Pa III - Chap e 3 a high-pe o mance liquid
ch oma og aphy (HPLC) me hod was de eloped and op imized o be used h oughou he
expe imen al wo k o his hesis and o allow he bes p ecise quan i ica ion o an ioxidan
con en in he na u al ex ac s, he nanopa icles associa ion e iciency and ei he elease
and pe meabili y p o iles, de eloped in he ollowing chap e s. In Pa IV - Chap e 4 a
comp ehensi e de elopmen , op imiza ion and physical-chemical cha ac e iza ion o
an ioxidan -chi osan nanopa icles was p esen ed. The e ec o osma inic acid con en ,
mass co ela ion and pH o nanopa icle p epa a ion we e e alua ed o he ionic gela ion
op imiza ion p ocess. Complemen a y me hodologies we e employed o p o ide a mo e
a ional unde s anding o he in e ac ions be ween componen s and he success o he
encapsula ion, such as he pa icle size and ze a po en ial. In Chap e 5 he nanoca ie s
we e e alua ed and cha ac e ized ega ding he in i o an ioxidan ac i i y po en ial. In
Pa V - Chap e 6 he nanoca ie s we e hen es ed o gua an ee hei sa e y
pe o mance, mucoadhesion p op ie ies and in i o ocula cell pe meabili y. In Chap e 7,
i was pe o med he i s a emp ing e o s o p o e osma inic acid he apeu ical po en ial
in an ischemia- epe usion (I-R) animal model. Finally, in Pa VI, he o e all conclusions
we e p esen ed in Chap e 8 - and u u e p ospec s, based on c i ical ques ions a ising
om his disse a ion, we e pu o wa d in Chap e 9.
xx
Mos in o ma ion p esen ed in he 9 chap e s ha cons i u e his disse a ion has been
al eady submi ed o in e na ional pee e iew, ia publica ion in scien i ic jou nals –
acco ding o he ollowing lis :
Pa I:
Chap e 1 – S a e o he a
Bap is a da Sil a S., Cos a J., Pin ado M., Fe ei a D., Sa men o B. (2010). An ioxidan s in
he p e en ion and ea men o diabe ic e inopa hy – A Re iew. Jo nal o Diabe es and
Me abolism 1:111. doi:10.4172/2155-6156.1000111.
Bap is a da Sil a S., Fe nandes J., Ta i a F., Pin ado M., Sa men o B. (2011). The
po en ial o chi osan in d ug deli e y sys ems. In Focus on Chi osan Resea ch, Edi ed by
A hu N. Fe guson and Amy G. O'Neill, No a Publishe s, ISBN: 978-1-61324-454-8.
Ta a ia, F., Fe nandes, J., San os-Sil a, A., Bap is a da Sil a, S., Sa men o, B. and
Pin ado, M. (2011). Biological ac i i ies o chi in, chi osan and espec i e oligome s. In
Focus on Chi osan Resea ch, Edi ed by A hu N. Fe guson and Amy G. O'Neill, No a
Publishe s, ISBN: 978-1-61324-454-8.
And ade F., An unes F., Nascimen o V., Ba is a da Sil a S., Ne es J., Fe ei a D.,
Sa men o B. (2011). Chi osan o mula ions as ca ie s o he apeu ic p o eins. Cu en
D ug Disco e y Technologies. 8(3):157-172. doi: 10.2174/157016311796799035.
Sa men o B., And ade F., Bap is a da Sil a S., Rod igues F., Ne es J., Fe ei a D. (2012).
Cell-based in i o models o p edic ing d ug pe meabili y. Expe Opinion on D ug
Me abolism and Toxicology. 8(5):607-621. doi: 10.1517/17425255.2012.673586.
Sil a N., Bap is a da Sil a S., Sa men o B., Pin ado M. (2013). Chi osan nanopa icles o
dap omycin deli e y in ocula ea men o bac e ial endoph halmi is. D ug Deli e y, doi:
10.3109/10717544.2013.858195.
Bap is a da Sil a S., Bo ges S., Ramos O., Pin ado M., Fe ei a D., Sa men o B. (2014).
T ea ing e inopa hies: Nano echnology as a ool in p o ec ing an ioxidan s agen s in
xxi
Sys ems Biology o F ee Radicals and An ioxidan s. Sp inge -Ve lag (Ge many), ISBN:
978-3-642-30017-2.
Bap is a da Sil a S., Bo ges S., Pin ado M., Sa men o B. (2014). Fo mula ion o essen ial
oils in pha maceu ical dosage o ms - biopha maceu ics and he apeu ic po en ials,
Pha maceu ical Biology. (Accep ed o publica ion).
Vasconcelos T., Bap is a da Sil a S., Fe ei a D., Pin ado M., Ma ques S. (2015). Cell-
based in i o models o ocula pe meabili y s udies. In Concep s and Models o D ug
Pe meabili y S udies: Cell and Tissue-based in i o Cul u e Models. Edi ed by B uno
Sa men o, Else ie . (Accep ed o publica ion).
Pa III:
Chap e 3 - High-pe o mance liquid ch oma og aphic me hod alida ion;
Bap is a da Sil a S., Oli ei a A., Fe ei a D., Sa men o B., Pin ado M. (2013).
De elopmen and alida ion me hod o simul aneous quan i ica ion o phenolic
compounds in na u al ex ac s and nanosys ems. Phy ochemical Analysis. 24(6): 638-644.
doi: 10.1002/pca.2446.
Pa IV:
Chap e 4 - De elopmen , op imiza ion and physical-chemical
cha ac e iza ion o chi osan-based nanopa icles;
Chap e 5 - In i o assessmen o an ioxidan ac i i y o chi osan-based
nanopa icles;
Bap is a da Sil a S., Amo im M., Fon e P., Madu ei a R., Fe ei a D., Pin ado M.,
Sa men o B. (2015). Na u al ex ac s in o chi osan nanoca ie s o osma inic acid d ug
deli e y. Pha maceu ical Biology. doi:10.3109/13880209.2014.935949.

xxii
Pa V:
Chap e 6 - In i o e alua ion o cy o oxici y, mucoadhesion and ocula
pe meabili y o osma inic acid in o chi osan-based nanopa icles;
Chap e 7 - The apeu ical po en ial e alua ion in ischemia- epe usion animal
model o chi osan based-nanopa icles;
Bap is a da Sil a S., Fe ei a D., Pin ado M., Sa men o B. E alua ion o chi osan-based
nanopa icles o ocula deli e y o osma inic acid h ough in i o mucoadhesion and
pe meabili y s udies, submi ed o publica ion.
xxiii
Lis o con en s
DECLARATION ............................................................................................................... IV
INSPIRATION ................................................................................................................... V
DEDICATION ................................................................................................................... VI
ACKNOWLEDGEMENTS ............................................................................................... VII
ABSTRACT ..................................................................................................................... XI
RESUMO ....................................................................................................................... XV
SCOPE AND OUTLINE ................................................................................................ XIX
LIST OF FIGURES .................................................................................................... XXVIII
LIST OF TABLES ....................................................................................................... XXXI
LIST OF ABBREVIATIONS ....................................................................................... XXXII
PART I - INTRODUCTION ................................................................................................ 1
CHAPTER 1 - STATE OF ART ........................................................................................... 3
1. INTRODUCTION ........................................................................................................ 5
2. CHITOSAN PROPRIETIES AND BIOMEDICAL APPLICATION ............................... 8
2.1. CHITOSAN-BASED DRUG DELIVERY SYSTEMS ............................................. 10
2.1.1. CHITOSAN SOLUTIONS ..................................................................................... 11
2.1.2. FILMS ............................................................................................................. 12
2.1.3. TABLETS ......................................................................................................... 14
2.1.4. HYDROGELS.................................................................................................... 16
2.1.5. MICROPARTICLES ............................................................................................ 19
2.1.6. NANOPARTICLES ............................................................................................. 21
2.2. CLINICAL TRIAL - SAFETY AND TOLERABILITY OF CHITOSA-N-ACETYLCYSTEINE EYE
DROPS IN HEALTHY YOUNG VOLUNTEERS ........................................................................ 23
3. OXIDATIVE PRODUCTS AND THE CLINICAL IMPORTANCE OF ANTIOXIDANTS
24
3.1.1. TYPES OF ANTIOXIDANTS AGENTS ..................................................................... 25
3.1.2. PHYSIOLOGY AND PATHOBIOLOGY OF REACTIVE OXYGEN SPECIES IN
RETINOPATHIES ............................................................................................................. 27
xxi
3.1.3. OXIDATIVE STRESS IMBALANCE AND RETINAL AFFECTED DISEASES ..................... 29
4. NANOTECHNOLOGY APPLIED TO ANTIOXIDANTS PROTECTION .................... 30
4.1. NANOANTIOXIDANTS PHARMACOTHERAPY ............................................................ 35
4.2. SAFETY ISSUES OF ANTIOXIDANT NANOPARTICLES ................................................ 36
5. SUMMARY .............................................................................................................. 38
PART II - AIMS AND GOALS ......................................................................................... 39
CHAPTER 2 - AIMS AND ORGANIZATION OF THE THESIS .................................................. 41
PART III .......................................................................................................................... 45
ABSTRACT .................................................................................................................... 47
CHAPTER 3 - HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD VALIDATION ....... 49
3. INTRODUCTION ...................................................................................................... 51
3.1. EXPERIMENTAL.................................................................................................. 52
3.1.1. MATERIALS ..................................................................................................... 52
3.1.2. EQUIPMENT AND CHROMATOGRAPHIC CONDITIONS ............................................ 52
3.1.3. PREPARATION OF STANDARD AND SAMPLE SOLUTIONS ....................................... 53
3.1.4. METHOD VALIDATION ....................................................................................... 53
3.1.5. METHOD APPLICABILITY ................................................................................... 54
3.2. RESULTS AND DISCUSSION ............................................................................. 54
3.2.1. APPLICATION OF THE CHROMATOGRAPHIC METHOD ........................................... 54
3.2.2. LINEARITY ....................................................................................................... 55
3.2.3. PRECISION ...................................................................................................... 56
3.2.4. ACCURACY ..................................................................................................... 58
3.2.5. SPECIFICITY .................................................................................................... 58
3.2.6. RANGE ........................................................................................................... 59
3.2.7. ROBUSTNESS .................................................................................................. 59
3.2.8. DETECTION LIMIT AND QUANTITATION LIMIT ....................................................... 59
3.2.9. METHOD APPLICABILITY ................................................................................... 60
3.3. CONCLUSION ..................................................................................................... 61
PART IV .......................................................................................................................... 63
ABSTRACT .................................................................................................................... 65
xx
CHAPTER 4 - DEVELOPMENT, OPTIMIZATION AND PHYSICAL-CHEMICAL CHARACTERIZATION
OF CHITOSAN-BASED NANOPARTICLES ............................................................................ 67
4. INTRODUCTION ...................................................................................................... 69
4.1. EXPERIMENTAL.................................................................................................. 70
4.1.1. MATERIALS ..................................................................................................... 70
4.1.2. PREPARATION OF CHITOSAN-BASED NANOPARTICLES ........................................ 70
4.1.3. ENCAPSULATION OF SAGE, SAVORY AND ROSMARINIC ACID INTO CHITOSAN-BASED
NANOPARTICLES ............................................................................................................ 71
4.1.4. SIZE AND SURFACE CHARGE ............................................................................. 71
4.1.5. MORPHOLOGY ................................................................................................. 72
4.1.6. ASSOCIATION EFFICIENCY ................................................................................ 72
4.1.7. IN VITRO RELEASE OF ROSMARINIC ACID FROM CHITOSAN NANOPARTICLES ......... 73
4.1.8. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY ANALYSIS AND ROSMARINIC ACID
QUANTIFICATION ............................................................................................................ 73
4.1.9. DIFFERENTIAL SCANNING CALORIMETRY ANALYSIS ............................................. 74
4.1.10. FOURIER-TRANSFORM INFRARED ANALYSIS ....................................................... 74
4.1.11. STATISTICAL ANALYSIS .................................................................................... 75
4.2. RESULTS AND DISCUSSION ............................................................................. 75
4.2.1. PARTICLE SIZE, POLYDISPERSITY AND ZETA POTENTIAL ...................................... 75
4.2.2. MORPHOLOGY ................................................................................................. 77
4.2.3. ASSOCIATION EFFICIENCY AND DRUG LOADING .................................................. 79
4.2.4. IN VITRO ROSMARINIC ACID RELEASE FROM CHITOSAN NANOPARTICLES .............. 80
4.2.5. THERMAL BEHAVIOR BY DIFFERENTIAL SCANNING CALORIMETRY ANALYSIS .......... 82
4.2.6. SPECTROSCOPY BY FOURIER-TRANSFORM INFRARED ANALYSIS ......................... 85
4.3. CONCLUSION ..................................................................................................... 88
CHAPTER 5 – IN VITRO ASSESSMENT OF ANTIOXIDANT ACTIVITY OF CHITOSAN-BASED
NANOPARTICLES ............................................................................................................ 89
5. INTRODUCTION ...................................................................................................... 91
5.1. EXPERIMENTAL.................................................................................................. 91
5.1.1. MATERIALS ..................................................................................................... 91
5.1.2. SAMPLE PREPARATION .................................................................................... 92
5.1.3. CHITOSAN NANOPARTICLES DEVELOPMENT AND OPTIMIZATION ........................... 92
xxxii
Lis o abb e ia ions
ABTS - 2,2-Azinobis (3-E hylbenzo hiazoline-6-Sulphonic) Acid
AE - Associa ion E iciency
AGE - Ad anced Glyca ion End P oduc s
AMD - Age Macula Degene a ion
ANOVA - One-Way Analysis o Va iance
ARVO - Associa ion o Resea ch in Vision and Oph halmology
BAB - Blood-Aqueous Ba ie
BRB - Blood-Re inal Ba ie
CAT - Ca alase
CBQF - Cen o de Bio ecnologia e Química Fina
Cuppe - Cu
Da - Dal on
DAPI - 4',6-Diamidino-2-Phenylindole
DD - Deace yla ion Deg ee
DL - De ec ion Limi
DMEM - Dulbecco’s Modi ied Eagle’s Medium
DMSO - Dime hyl Sul oxide
DNA - Desoxy ibonucleic Acid
DPPH - Diphenyl-1-Pic ylhyd azyl
DSC - Di e en ial Scanning Calo ime y
EMA - Eu opean Medicines Agency
ERG - Elec o e inog ams
FDA - Food and D ug Adminis a ion
FFUP - Faculdade de Fa mácia da Uni e sidade do Po o
FTIR - Fou ie T ans o m In a ed
GCL - Ganglion Cell Laye
GNP - Gold Nanopa icles
GPx - Glu a hione pe oxidase
GSH - Glu a hione
HBA - p – Hyd oxybenzyl Alcohol
HBSS - Hanks’ Balanced Sal Solu ion
HCE-T - Human Co nea Cell Line
HET-CAM - Cho ioallan oic Memb ane Tes

xxxiii
HPLC - High Pe o mance Liquid Ch oma og aphy
HPOX - Hyd oxybenzyl Alcohol Inco po a ed Copolyoxala e
IBILI - Ins i u e o Biomedical Imaging and Li e Sciences
INEB - Ins i u o de Engenha ia Biomédica
INL - Inne Nuclea Laye
IOBA – NHC - Immo alized Epi helial Cell Line om Human Conjunc i a
IOP - In aocula P essu e
IPL - Inne Plexi o m Laye
I-R - Ischemia-Repe usion
ISCS-N - Ins i u o Supe io de Ciências da Saúde – No e
LCPUFA - Long-Chain Polyunsa u a ed Fa y Acid
LDH - Lac a e Dehyd ogenase
MNP - Magne ically Responsi e Nanopa icles
MnSOD - Supe oxide Dismu ase
MSc - Mas e o Science
MTT - Thiazolyl Blue Te azolium B omide
MW - Molecula Weigh
NR - Rosma inic Acid Nanopa icles
NSG - Sa u eja mon ana Nanopa icles
NSV - Sal ia o icinalis Nanopa icles
OCT - Op imal Cu ing Tempe a u e
ORAC - Oxygen Radical Abso bance Capaci y
OS - Oxida i e S ess
Papp - Appa en Pe meabili y Coe icien
PBS - Phospha e Bu e Saline
PdI - Polydispe si y Index
PhD - Philosophiæ Doc o
PLA - Polylac ic Acid
PP - Polyp opylene
PUFA - Polyunsa u a ed Fa y Acid
PVA - Poly (Vinyl Alcohol)
QL - Quan i ica ion Limi
QUEN - Que ce in Nanopa icles
RGC - Re inal Ganglion Cells
ROS - Reac i e Oxygen Species
xxxi
RPE - Re ina Pigmen Epi helium
pm - Ro a ions pe Minu e
RSD - Rela i e S anda d De ia ion
SD - S anda d De ia ion
SEM - Scanning Elec on Mic oscopy
SiNPs - Silica e Nanopa icles
SOD - Supe oxide dismu ase
TEM - T ansmission Elec on Mic oscopy
TPP - T ipolyphospha e
TUNNEL - Te minal Deoxynucleo idyl T ans e ase (TdT)-Media ed dUTP Nick End
Labeling
UV - Ul a-Viole
- Volume
VEGF - Vascula Endo helial G ow h Fac o
w - Weigh
Zn - Zinc
1
PART I - In oduc ion
“Somewhe e, some hing inc edible is wai ing o be known”
Ca l Sagan
2
3
CHAPTER 1 - S a e o a

4
5
1. In oduc ion
In pha maceu ical science he e is a con inuous blockbus e d ug de elopmen , and
nowadays biomolecules as ac i e agen s, a e widely explo ed o de elop new
he apeu ics. Ne e heless, mos o hese new ac i e compounds a e uns able and mus
be p o ec ed om deg ada ion in he physiological en i onmen , due o he poo
abso p ion ha cons ains he anspo ac oss biological ba ie s. Thus, he e icacy o
mos d ugs clea ly depends on he design o app op ia e ca ie s o hei physical
p o ec ion, deli e y and con olled elease (1). Among he di e en app oaches explo ed
so a , colloidal ca ie s a e pa icula ly in e es ing, especially hose made o
mucoadhesi e polyme s o assu e hei epi helium pe manence (2, 3). Fo his applica ion,
chi osan has had qui e impac in he associa ion and deli e y o labile mac omolecula
compounds (4). Chi osan ca ie s ha e an excep ional po en ial o d ug deli e y,
especially o mucosal, since hese sys ems a e s able in con ac wi h physiological luids
and ba ie s. They a e also able o con ol d ug elease and p o ec agains ad e se
condi ions like mucosal enzymes and biological p o ec i e luids. Due o i s
mucoadhesion, pa icle size, pa icle su ace chemis ies, cha ge and he unique
abso p ion enhancing p ope ies, he chi osan po en ial in he medical ield is widely
p omising. Di e en o mula ions such as ilms, able s, hyd ogels, mic o and
nanosys ems a e expec ed o op imize, cha ac e ize and selec he d ug pe o mance,
imp o ed p ope ies and inc ease s abili y o g ea speci ic applica ions.
Pha macokine ics and oxicological ele ance o chi osan sys ems a e gua an eed by in
i o model sys ems in molecula , subcellula and cellula le els, as well as hei
he apeu ic e icacy and sa e y pe o mance should also be p o en in i o. One ca ego y
o compounds in which hese chi osan ca ie s may be a key o success, a e he
an ioxidan s. Conside ing he biology de ini ion, an ioxidan s a e chemical compounds o a
subs ance ha inhibi s oxida ion, coun e ac ing he damage o ee adicals e ec s in a
li ing o ganism, and o his eason a e eac i e species (5). An ioxidan s a e widesp ead
i ually in plan oods, o en a high le els, and include phenols, phenolic acids and
la onoids (6). Rosma inic acid (a-O-ca eoyl-3,4-dihyd oxyphenillac ic acid) (7) is a
phenolic compound, which can p o ide p o ec ion agains cance (7) and ha e o he
mul i ude biological ac i i ies, namely ads ingen , an i-in lamma o y, an i-mu agen,
an ibac e ial and an i i al (7, 8). The la e ac i i y has been es ed in he he apy o
He pes simplex in ec ions wi h osma inic acid-con aining ex ac s o Melissa o icinalis
(7). I is also one o he e icien na u al an ioxidan s (9) since osma inic acid displays a
6
huge po en ial adical sca enging ac i i y, highe han olox (a de i a i e o a- ocophe ol)
(10-12). Rosma inic acid has also an an i-angiogenic ac i i y o e inal neo ascula iza ion
in a mouse model o e inopa hy (13). Signi ican ly inhibi ed he p oli e a ion o e inal
endo helial cells in a dose-dependen manne , and inhibi ed in i o angiogenesis o ube
o ma ion. Mo eo e , osma inic acid showed no e inal oxici y. These da a sugges
osma inic acid could be a po en inhibi o o e inal neo ascula iza ion and may be
applied in he ea men o asop oli e a i e e inopa hies (13). I is he majo componen
o Sal ia o icinalis (sage) and Sa u eja mon ana (sa o y) na u al ex ac s. These a e
plan s o en used in adi ional medicine, and which g ow in he poo soils o he
Medi e anean basin (14). Besides applica ion as condimen , sage and sa o y ha e been
used as an an i-dia hea ec o , diges ion adju an , con ibu e o heal wounds, play an
an i-in lamma o y ole, disin ec an , igh insomnia and dec ease blood p essu e. Some o
hese biological ac i i ies ha e been associa ed wi h i s high con en s o osma inic acid
and he p esence o o he ele an phenolic compounds such as que ce in and u in (9,
14). Beyond he biological huge bene i s, an ioxidan s a e ex emely sensi i e o ligh ,
oxygen, a e highly eac i e wi h o he compounds, in some cases possess poo solubili y,
ine icien pe meabili y, and a e ex emely uns able (15-17). Fo all hese easons hei
deli e y using he con en ional dosage o ms is a challenge (18). In his con ex ,
al e na i e ca ie s a e being conside ed, ega ding he op imiza ion o pha macokine ics
and pha macodynamics o an ioxidan molecules. Chi osan nanopa icles, due o hei
p ope p ope ies, a e on he aw. Fo his conce n he nano echnology is expec ed o
inc ease he abili y, o e ain he an ioxidan ac i i y du ing he p epa a ion p ocess, o
op imize he elease o he compound om he ca ie sys em, and o ensu e a good
con ol o hei physical-chemical p ope ies inc easing hei s abili y. I hese nanoca ie s
may imp o e he e icacy pe o mance o he an ioxidan s, se e al diseases like cance ,
diabe es, hype ension, a e io-scle osis, ca dio ascula disease o ocula anomalous
condi ions ha ha e a clinical impai men wi h oxida ion p ocesses, may be p e en ed o
be e con olled (19). Conside ing he eye diso de s, he e a e many ypes o
e inopa hies condi ions ha may ha e an oxida i e e iology (20, 21), like e ini is
pigmen osa, glaucoma, macula degene a ion, e inoblas oma and diabe ic e inopa hy
(19, 21). Mul iple ac o s ha e been also p oposed o explain e inopa hies, including
gene ic diso de s, in ec ions by mic obial agen s, so bi ol pa hway hype ac i i y,
accumula ion o ad anced glyca ion end p oduc s (AGEs) (22) and p o ein kinase C
ac i a ion (23). Ne e heless he p ecise pa hological mechanism emains o be
elucida ed. Which is clea is he colla e al damage o hese diso de s, ha may esul on
7
e lec i i y changes, bi u ca ions, o uosi y neo ascula iza ion as well as o he pa e ns o
blood essels and e en blindness (19, 21). In he case o ocula pa hologies he oxida i e
s ess (OS) clinical impai men has a signi ican impac , since he ocula globe is he
o gan mos a ec ed by OS. I s cons an ly expose o ligh and oxygen and i s high
polyunsa u a ed a y acid (PUFA) con en ha is p one o lipid pe oxida ion, may be some
p ominen easons (24). OS is also associa ed wi h inc eased ascula pe meabili y,
dis up ion o blood- e inal ba ie , apop o ic loss o e inal capilla y cells, mic o ascula
abno mali ies and neo ascula iza ion (25). High le els o OS a e also usually associa ed
wi h inc eased le els o oxida i e modi ied desoxy ibonucleic acid (DNA) and ni osyla ed
p o eins, and an ioxidan de ense enzymes impai (26). When such damages a e
p esen ed, wi hou e ec i e medical ea men , cells and issues o he e ina become
malnou ished and p og essi ely degene a e, which leads o damage in cells esponsible
o ision, leading o i s ine i able loss (21). Due o his in ima e ela ionship be ween OS
and he pa hogenesis o e inopa hies, he use o app op ia e an ioxidan s may ha e
po en ial on he me abolic and unc ional abno mali ies in e inopa hies (27). Ne e heless,
o an ioxidan s assu e hese condi ions hey need he nanoca ie suppo o ake hem o
he igh place wi hou losing hei unc ional ac i i y.
14
p o ide al e na i e me hods o ea men o clinicians o comp omised wound si es whe e
a ascula zones can p e en he deli e y o an ibio ics o he in ec ed issue. A ecen
s udy demons a ed ha inco po a ing an ibio ics in chi osan ilms could p o ide
al e na i e me hods o ea ing musculoskele al in ec ions (60).
No el chi osan based polyelec oly e complexes we e de eloped and op imized in o de o
ob ain ilms possessing he op imal unc ional p ope ies ( lexibili y, esis ance, wa e
apo ansmission a e and bioadhesion) o be applied on skin (61). The de elopmen
was based on he combina ion o chi osan and wo polyac ylic acid polyme s wi h di e en
c oss-linke s and c osslinking densi ies. The op imized ilm, including adhesi e p ope y,
has shown e y good p ope ies o applica ion in he skin and ep esen s a e y
p omising o mula ion o u he inco po a ion o d ugs o opical and ansde mal
adminis a ion.
2.1.3. Table s
Va ious s udies wi h chi osan ega ding con olled elease deli e y sys ems ha e been
conduc ed o o al dosage o ms, om ilm coa ed pelle s, able s o capsules o mo e
sophis ica ed and complica ed deli e y sys ems such as osmo ically d i en sys ems,
sys ems con olled by ion exchange mechanism, sys ems using h ee dimensional p in ing
echnology and sys ems using elec os a ic deposi ion echnology (62). The mos common
con olled deli e y sys em ha e been able s and g anules because o i s e ec i eness,
low cos , ease o manu ac u ing and p olonged deli e y ime pe iod, whe e he d ug is
uni o mly dissol ed o dispe sed h oughou he polyme (62). The able ing p ocess is
associa ed wi h ela i ely high p essu e in o de o o m sui able compac s. Howe e , no
only he able ing excipien s a e de o med du ing he p ocess o able o ma ion, bu also
he able i sel . This can lead o o al o pa ial damage o such ma e ials, namely loss o
biological ac i i y o p o eins and enzymes, polymo phic ans o ma ion o excipien s o
damage o he coa ing ma e ial. Mos ecen ly, di e en excipien s we e es ed in o de o
p e en such damages. Amongs o he s, polysaccha ides like chi osan and ca ageenan
ha e shown o be ad an ageous because o hei elas ic able ing beha io (63). Se e al
epo s ha e been published on he use o chi osan as able excipien s. I was applied as
a ca ie o sus ained elease able s, a di ec comp essible diluen , a able disin eg a e
and a able binde (37). As a diluen , chi osan was used o p epa a ion o di ec
comp essed able s (64, 65) whe e d ug elease was con olled. S udies using chi osan as

15
di ec ly comp essible able excipien showed i s po en ial o use in modi ied elease d ug
deli e y sys ems wi hou he need o addi ional adju an s (64). Chi osan also showed
highe binde e iciency han o he able binde s such as me hylcellulose and sodium
ca boxyme hylcellulose (66) and used as a binde o colon speci ic d ug deli e y able s
wi h slow d ug elease compa ed wi h o he polysaccha ides o syn he ic polyme s (67).
Chi osan was u ilized as able s disin eg a e (66) and showed bioadhesi e p ope ies in
mix u e wi h sodium algina e and in he o m o hiola ed chi osan de i a i e wi h slow d ug
elease o in a-o al d ug deli e y able s (68). Fu he mo e, he solubilizing and
amo phizing p ope ies o low MW chi osan owa d nap oxin made i an op imal ca ie o
de eloping as elease o al able (69). Depending mainly on ionic in e ac ion, chi osan
was also used o he p epa a ion o able s ma ix o con ol d ug elease (70, 71).
When used in a ma ix- ype able o mula ion, chi osan o ms a gel-ba ie in an acid
en i onmen ha can modula e o cons ain d ug elease. Fu he mo e, a acidic pH
amines o chi osan a e p o ona ed and can he e o e in e ac wi h opposi ely cha ged d ug
ions, se ing as excipien o modi ied elease o d ug deli e y sys ems (65).
Chi osan was s udied as excipien in he p epa a ion o p olonged heophylline able s.
These able s showed highe d ug bioa ailabili y han o he comme cial ones, which
becomes a new po en ial o mula ion o espi a o y p oblems (66).
The biological po en ial o chi osan adds also clea bene i s o he able o mula ion and
p ocess. In a ecen esea ch i e di e en polysaccha ides wi h po en ial an ioxidan
ac i i y o ex ended- elease ma ix able s we e compa ed (72). The esul s sugges ha
chi osan would be po en ially use ul in an ex ended- elease able wi h he highe
an ioxidan ac i i y, able o ca ch he mos di e se and na u al oxida i e species, usually
in ol ed in di e en pa hologies.
A new s udy conce ning aginal in ec ions and in lamma ions we e e alua ed using
chi osan able s (73). Topical adminis a ion o he an ibac e ial me onidazole ep esen s
he mos common he apy in he ea men o bac e ial aginosis caused by T ychomonas
aginalis. The o mula ions gene ally a ailable o such he apy a e c eams, gels, aginal
la ages and aginal supposi o ies. In his s udy, a new dosage o m, con aining
me onidazole was de eloped wi h he aim o ealize aginal mucoadhesi e able s by
including bioadhesi e polyme s as chi osan. This kind o deli e y sys ems sui able o
o mula ing me onidazole o opical applica ion ep esen s a good al e na i e o
adi ional dosage o ms o aginal opical adminis a ion in he ea men o in ec ions o
in lamma ions. These solu ions o e lap he limi a ions o con en ional he apies ha a e
16
no sui able o assu e d ug pe manence on he aginal mucosa su ace o adequa e ime
assu ing he comple e elimina ion o bac e ia and pa hology e adica ion.
None heless, all applica ions o chi osan as able excipien we e no in i s de i a i e
o ms. Howe e , a emp s ha e been made o imp o e chi osan p ope y by de eloping
de i a i e sal s. Chi osan de i a i es such as glu ama e, aspa a e and hyd ochlo ide sal s
ha e been used o colon-speci ic d ug deli e y and o enhance he deli e y o he apeu ic
pep ide ac oss in es inal epi helia (37).
2.1.4. Hyd ogels
Hyd ogels a e ne wo ks o hyd ophilic polyme s ha can abso b la ge quan i ies o wa e
wi hou dissolu ion. Due o hei physical p ope ies esembling human issue and i s
excellen issue compa ibili y, hyd ogels ha e been ex ensi ely s udied o biomedical
applica ions. They can be used as so con ac lenses (74), issue enginee ing sca olds
(75), d ugs ca ie s and con olled- elease sys ems (76). In addi ion, hyd ogels ha e he
po en ial o u he healing (77). They can abso b excess wound exuda es, p o ec he
wound om seconda y in ec ion and e ec i ely p omo e he healing p ocess by p o iding
an en i onmen o mois wound healing (78). E en can also be emo ed wi hou causing
auma o he wound (78).
Se e al models o hyd ogels ha e been s udied, including chi osan, poly ( inyl alcohol)
(PVA) ha is a wa e -soluble polyhyd oxy polyme and algina e. Howe e , chi osan has
been widely exploi ed in hyd ogel o mula ion and in p ac ical applica ions because o i s
easy manipula ion, excellen chemical esis ance, physical p ope ies, biodeg adabili y
and low p ice (78). This polyme is used o p oduce hyd ogels wi h well-known p ope ies
ha a e used o deli e y o p o eins and syn he ic d ugs. Since his compound is also
polyelec oly e, i s ionic o m p oduces complexes h ough hyd ogen bonding o
elec os a ic in e ac ions. Besides his, ano he in e es ing p ope y o chi osan is i s abili y
o gel in con ac wi h speci ic polyanions. This gela ion p ocess is due o he o ma ion o
in e - and in amolecula bonding media ed by hese polyanions (1). In he las decade,
di e en chi osan hyd ogels we e p oduced o d ug deli e y in mic o o nano-scale using
he polyelec oly e complexa ion echnique. The e a e many ac o s ha a ec he ele an
p ope ies o he capsules o chi osan, in pa icula he composi ion, MW and DD o
chi osan. Se e al me hods ha e been de eloped in which he pa icle size o chi osan
17
hyd ogel and i s ela ed p ope ies a e qui e dis inc , acco ding o he me hod o
p epa a ion and he eac ion condi ions ha a e employed. One o he majo ac o s ha
may in luence he inal p ope ies is he me hod o p epa ing hyd ogel. Mo eo e , ew
a emp s we e made o co ela e s a is ically he eac ion condi ions wi h he inal
p ope ies o chi osan hyd ogel. Liu and his collabo a o s (79) e alua ed he in luence o
chi osan MW and i s concen a ion, along wi h pH, upon he swelling beha io o
mic ocapsules o chi osan-algina e, and pos ula ed ha all ac o s ha e an e ec on he
beha io o he hyd ogel swelling. The algina e-chi osan hyd ogels a e commonly
p epa ed by ionic complexa ion using algina e as a gel co e (80) and hen cha ac e ized
by he ib a ion modes o hei main g oups using FTIR. O he hyd ogels o mula ion
p ocedu es can be pe o med by UV c osslinking. In his me hod, lac ose moie ies a e
in oduced in o chi osan o ob ain much be e wa e -soluble chi osan a neu al pH, and
pho o eac i e azide g oups a e added o p o ide he abili y o o m a gel h ough
c osslinking azide g oups wi h amino g oups (81). This pho oc osslinkable chi osan is hen
exposed o UV i adia ion o o m an insoluble and adhesi e hyd ogel wi hin 60 s.
Hyd ogel has he consis ency o anspa en and so ubbe (81).
The c osslinking can also be pe o med by high empe a u e. I is based on he
neu aliza ion o a chi osan solu ion wi h a polyol coun e ionic dibase sal such as β-
glyce ophospha e. Chi osan/glyce ophospha e is a he mosensi i e solu ion, which is
liquid a oom empe a u e and solidi ies in o a whi e hyd ogel a body empe a u e (81).
In addi ion, c osslinking can be achie ed by high pH, employing he pH-sensi i e p ope y
o chi osan solu ions a low pH. Once injec ed in o he body, hese polyme solu ions ace
di e en en i onmen al pH condi ions and o m gels (81).
The chi osan hyd ogel o mula ion can also be made by, eezing, hawing o chemical
me hods. I adia ion has he ad an ages o easy con ol o p ocessing, wi hou adding
ini ia o s o c oss-linke s ha can be ha m ul and di icul o emo e and also has he
op ion o combining he hyd ogel o ma ion and s e iliza ion in one echnological s ep. The
main disad an age o hyd ogels p epa ed by i adia ion is i s poo mechanical s eng h.
Howe e , hyd ogels p epa ed by eeze- haw o example, o aqueous solu ions o PVA
has good mechanical s eng h, a e s able a oom empe a u e and does no equi e
ini ia o s o c oss-linke s. The main disad an age o his ype o hyd ogel is i s opaque
appea ance and limi ed expansion capabili y (78).
The hyd ogel yields a e e alua ed h ough he weigh di e ence, placing he washed
hyd ogels in o p e-weighed lasks and hen in o a s o e a 50 ºC un il d yness. The
chi osan hyd ogel pa icles can hen be isualized and cha ac e ized by pa icle size and
18
size dis ibu ion using an in e ed op ical mic oscope (80) as well as, by solubili y, X- ay
di ac ion, he mal analysis, and sol en up ake (82).
An oph halmic deli e y sys em wi h imp o ed mechanical and mucoadhesi e p ope ies
ha could p o ide p olonged e en ion ime o he ea men o ocula diseases we e
e alua ed conside ing chi osan hyd ogel o mula ion. Fo his, an in si u o ming gel was
de eloped by he combina ion o a he mose ing polyme , poly (e hylene oxide)–poly
(p opylene oxide)–poly (e hylene oxide) wi h chi osan. The e o e, he inal o mula ion
p esen ed adequa e mechanical and senso y p ope ies and emained in con ac wi h he
eye su ace o a p olonged ime. In conclusion, he in si u o ming gel comp ised o
poloxame /chi osan is a p omising ool o he opical ea men o ocula diseases (83).
To o e lap he limi a ion o opical deli e y o an imic obial agen s and o p olong ac i e
d ug concen a ions in he o al ca i y, i was designed a hyd ogel o mula ion con aining
chi osan o deli e y o chlo hexidine glucona e o he o al ca i y (84). Chi osan p olongs
he adhesion ime o o al gels and d ug elease also inhibi ing he adhesion o Candida
albicans o human buccal cells since i has an i ungal ac i i y (84). The an i ungal agen ,
chlo hexidine glucona e also induces he educ ion o Candida albicans adhesion o o al
mucosal cells.
The p epa a ion and cha ac e iza ion o hiol-modi ied chi osan, which o med c osslinked
hyd ogels, was also desc ibed o cha ac e ize in i o elease kine ics o insulin
encapsula ed in di e en chi osan MW hyd ogels and e alua ed o hei po en ial use as a
sca old o he cul u e o NIH 3T3 cells (85). The esul s demons a ed ha insulin is no
immobilized locally wi hin he gel ne wo k. Since he inco po a ion in o he gel has no
impac on insulin s abili y i may be assumed ha he chi osan he mogelling sys em is an
a ac i e deli e y sys em o pep ides and p o eins.
The main goal o o he s udy was o de eloped a chi osan bioadhesi e gel o nasal
deli e y o insulin (86). The p oposed gel o mula ion could be use ul p epa a ion o
con olled deli e y o insulin h ough he nasal ou e and may ep esen an al e na i e
ea men o diabe es.
Mucoadhesi e chi osan lac a e gels we e de eloped in ended o he con olled elease o
lac ic acid on o aginal mucosa (87). The conclusions inding makes i easonable o
en isage a comple e elease o lac a e om he es ed o mula ions in aginal
en i onmen . A ecen esea ch epo s an in si u gelling chi osan-based hyd ogel sys em
ha sus ains he elease o a po en ial an i-cance gene (pigmen epi helium-de i ed
ac o ) o he umo si e. A signi ican educ ion o he p ima y os eosa coma in a clinically
ele an o ho opic model was measu ed. The combina ion o plasmid ea men and
19
chemo he apy oge he wi h he use o his deli e y sys em led o he highes supp ession
o umo g ow h wi hou side e ec s. The esul s ob ained om his s udy demons a e he
po en ial applica ion o a hyd ogel sys em as an an i-cance d ug deli e y o success ul
chemo-gene he apy (88).
Ano he no o ious s udy ocuses on he cu en use o injec able o o m in si u chi osan
hyd ogels in cance ea men (81). Fo mula ion p o ocols o in si u hyd ogel sys ems,
hei cy o oxic p ope ies, loading and in i o elease o d ugs, hei in i o e ec on cell
g ow h, he inhibi ion o umo g ow h in i o using mouse models, and u u e di ec ions o
enhance his echnology we e discussed. In conclusion, chi osan gelling sys ems due o
hei an ibac e ial, biocompa ible, biodeg adable and mucoadhesi e p ope ies a e a
po en ial ca ie o a ious cance ea men s.
These hyd ogels may also be use ul o de ec he localized g ow h o cells (81), which can
also be di ec ed o inno a i e me hods o diagnosis.
2.1.5. Mic opa icles
Mic opa icles a e de ined as mul ipa icula e deli e y sys ems, usually sphe ical wi h size
a ying om 1 o 1000 µm, con aining a co e ac i e subs ance (48). The e ms
mic ocapsules and mic osphe es a e o en used synonymously. Sphe es and sphe ical
pa icles a e also used o a la ge size and igid mo phology (48).
The use o mic opa icles-based he apy allows d ug elease o be ca e ully ailo ed o he
speci ic ea men si e h ough he choice and o mula ion o a ious d ug–polyme
combina ions. The o al dose o medica ion and he kine ics o elease a e he a iables,
which can be manipula ed o achie e he desi ed esul . Using inno a i e
mic oencapsula ion echnologies, and by a ying he copolyme a io, MW o he polyme
among o he pa ame e s, mic opa icles can be de eloped in o an op imal d ug deli e y
sys em, which will p o ide he desi ed elease p o ile (34).
Chi osan wi h di e en MW and concen a ion, deg ada ion a e o chi osan pa icles and
d ug concen a ion in e e e on mic opa icle p ope ies (1). Chi osan mic opa icles a e
used o p o ide con olled elease o many d ugs and o imp o e he bioa ailabili y o
deg adable subs ances such as p o ein o enhance he up ake o hyd ophilic subs ances
ac oss he epi helial laye s (34). Ha ing in mind bio/mucoadhesi e p ope ies o na u al
biopolyme s, chi osan mic opa icles ha e po en ial o colon a ge ing. In o de o achie e
localiza ion and p olonged esidence ime in he colon, ma ices should ha e op imal

20
pa icle size, be ween 4 and 15 µm (89). Ca ie sys ems in ha size ange a e able o
a ach mo e e icien ly o he mucus laye and accumula e in he a ec ed egion wi hou
he need o mac ophage up ake. This no el o mula ion will o e e icien ea men o
colon in lamma o y diseases like ulce a i e coli is and Ch on’s disease (89), inc easing
he apeu ic concen a ion (a he si e o in lamma ion) and ac i i y and minimizing side
e ec s ha occu by con en ional sys emic abso p ion. Chi osan mic opa icula e ca ie
sys ems a e also e icien in he ea men o in lamma o y bowel diseases (89).
Budesonide is one o he mos used d ug subs ances in he ea men o ac i e
in lamma o y bowel diseases. Chi osan mic opa icles loaded wi h budesonide we e
p oduced using no el one s ep sp ay-d ying p ocedu e. Coa ed mic opa icles we e
sui able candida es o o al deli e y o budesonide wi h con olled elease p ope ies o
local ea men o in lamma o y bowel diseases.
Chi osan mic opa icles also ep esen s a p omising polyme in nasal pep ide deli e y (90)
p olonging he esidence ime o nasal d ug deli e y sys ems a he si e o d ug
abso p ion. Addi ionally, chi osan imp o es he abso p ion o pep ides by opening
ansien ly he igh junc ions.
O al adminis a ion o he nons e oidal an i-es ogen amoxi en is he ea men o choice
o me as a ic es ogen ecep o -posi i e b eas cance . Chi osan mic opa icles we e
de eloped o amoxi en deli e y in o he lympha ic sys em (91), imp o ing amoxi en o al
bioa ailabili y and dec easing i s side e ec s. These da a unde line o he po en ial
he apies o his se ious cance condi ion.
I was also epo ed he impo ance o chi osan mic opa icles in he pu i ica ion o
immunoglobulin G om human plasma by a ini y ch oma og aphy using linoleic acid
a ached chi osan mic opa icles (92). I was concluded ha he mic opa icles allowed jus
one-s ep pu i ica ion o immunoglobulin G om human plasma.
Chi osan and i s de i a i e N- ime hyl chi osan chlo ide, gi en as mic opa icles
associa ed o he non- oxic mucosal adju an LTK63, we e e alua ed o in anasal
immuniza ion wi h he g oup C meningococcal conjuga ed accine. The bac e icidal
ac i i y measu ed in se um o mice immunized in anasally wi h he conjuga ed accine
o mula ed wi h his deli e y sys em and he LT mu an was supe io o he ac i i y in
se um o mice immunized sub-cu aneous. Impo an ly, in anasal bu no pa en e al
immuniza ion, induced bac e icidal an ibodies a he nasal le el, when o mula ed wi h
bo h deli e y sys em and adju an (93).
In ano he s udy, i was e alua ed he abili y o chi osan mic opa icles o enhance bo h
he sys emic and local immune esponses agains diph he ia oxoid a e o al and nasal
21
adminis a ion in mice. Signi ican sys emic humo al immune esponses we e also ound
a e nasal accina ion wi h diph he ia oxoid associa ed o chi osan mic opa icles.
Diph he ia oxoid associa ed o chi osan mic opa icles esul s in p o ec i e sys emic and
local immune esponse agains his oxoid a e o al accina ion and in signi ican
enhancemen o immunoglobulin G p oduc ion a e nasal adminis a ion. Hence, hese in
i o expe imen s demons a e ha chi osan mic opa icles a e e y p omising mucosal
accine deli e y sys em (94). O he simila s udies conside ed he chi osan mic opa icles
as encapsula ing agen o la ge amoun s o an igens such as o albumin, o e anus oxoid
(95). Besides chi osan pa icles a e a p omising candida e o mucosal accine deli e y,
mucosal accina ion no only educes cos s and inc eases pa ien compliance, bu also
limi s he in asion o pa hogens h ough mucosal si es.
2.1.6. Nanopa icles
Nanopa icles a e de ined as a mic oscopic pa icle whose size is in he nanoscale,
a ying om 1 o 1000 nm, able o deli e d ugs (2) o he igh place, a app op ia e imes
and a he igh dosage, also imp o ing hei bioa ailabili y, e iciency and educing
cy o oxici y associa ed o o he sys emic d ugs ca ie s, ac ually becomes one o he mos
a ac i e a eas o esea ch in d ug deli e y (2). These submic on pa icles con aining
en apped d ugs a e in ended o en e al o pa en e al adminis a ion, which may p e en
o minimize he d ug deg ada ion and me abolism as well as cellula e lux, ex ending he
shel -li e (50). Some esea che s ha e also obse ed ha he numbe o nanopa icles,
which c oss he in es inal epi helium is g ea e han ha o he mic opa icles and
hyd ophilic nanopa icles gene ally ha e longe esiden ime in blood hen mic osphe es
(96). Wi h hei easy accessibili y in he body, nanopa icles can also be anspo ed ia
he ci cula ion o di e en body si es. These pa icula e deli e y sys ems ha e been
shown o enhance he immune esponse ollowing mucosal applica ion. Nanopa icles
ha e been made o sa e ma e ials, including syn he ic biodeg adable polyme s, na u al
biopolyme s, lipids and polysaccha ides and ha e he po en ial o o e coming impo an
mucosal ba ie s, such as he in es inal, nasal and ocula ba ie s (50). Chi osan based
nanopa icles show g ea po en ial o deli e ing mac omolecula he apeu ics (in
pa icula d ugs and genes) and con ol he comple e elease o he d ugs in hei na i e
o ms ac oss biological ba ie s (41). Impo an ad an ages o hese nanopa icles include
22
hei apid p epa a ion unde ex emely mild condi ions and also hei abili y o inco po a e
bioac i e compounds (45). Chi osan nanopa icles opically applied in o he eye has been
p o en o inc ease he esidence ime o d ugs in he p eco neal a ea due o hei
adhesi e p ope ies and, he e o e, could p olong he pene a ion o d ugs in o he
in aocula s uc u es (2). An impo an esea ch shows ha a chi osan de i a i e can be
used o p epa e no can ha idin-associa ed nanopa icles by aking ad an age o he ionic
c oss-linkage be ween he d ug molecule and o he chi osan ca ie o an i-
hepa oca cinoma medicine (97).
The po en ial o nanopa icles as a accine deli e y has also been demons a ed in
se e al s udies (98-100). Nanopa icle-media ed gene deli e y is an al e na i e o i al
gene deli e y. Nanopa icles o e he po en ial o sa e, a ge ed and e icien gene
deli e y in a a ie y o o gans (101).
Bo ges e al. (102), ecen ly desc ibed a deli e y sys em ha is composed o a
nanopa icula e chi osan co e o which he hepa i is B su ace an igen (AgHBs) was
adso bed and subsequen ly coa ed wi h sodium algina e. The enhancemen o he
immune esponse obse ed wi h he an igen-loaded nanopa icles demons a ed ha
chi osan is a p omising pla o m o pa en e al deli e y o his an igen, since i esul ed in a
mixed Th1/Th2 ype immune esponse.
Chi osan nanopa icles ha e been also p oduced as a ca ie sys em o he nasal deli e y
o a mono alen in luenza subuni accine (103). The in anasal adminis e ed an igen-
chi osan nanopa icles induced highe immune esponses compa ed o he o he
in anasal an igen o mula ions, and hese esponses we e enhanced by in anasal
boos e accina ions. Mo eo e , among he es ed o mula ions only in anasal
adminis e ed an igen-con aining chi osan nanopa icles induced signi ican
immunoglobulin A le els in nasal washes o all mice es ed, demons a ing ha chi osan
nanopa icles a e a po en new deli e y sys em o in anasal adminis e ed in luenza
an igens.
Ano he s udy indica e ha chi osan nanopa icles a e a good ca ie o DNA accines
agains ube culosis by pulmona y deli e y, which may p o ide an ad an ageous deli e y
ou e compa ed o in amuscula immuniza ion, due inc easing highe immunogenici y
(104).
Chi osan nanopa icles ha e been also in oduced as a use ul ca ie o pep ide o al
deli e y, because hey can p o ec hese compounds om deg ada ion. Insulin, like o he
pep ides, has low he apeu ic ac i i y when adminis e ed o ally due o deg ada ion by
p o eoly ic enzymes (105).
23
Sa men o e al. (106) de eloped chi osan nanopa icles as d ug ca ie o insulin. Insulin
was en apped in di e en polyanion/chi osan nanopa icula e sys ems wi h high
e iciency, o s udy mo phologic and physical p ope ies o esul ing nanopa icula e
complexes and o in es iga e insulin elease beha io unde gas oin es inal condi ions
(106). These nanopa icula e complexes appea o possess good p ope ies o o al
p o ein deli e y, pa icula ly hose con aining dex an sul a e/chi osan polyelec oly es,
which p o ided highes insulin associa ion e iciency and e en ion o insulin in gas ic
simula ed condi ions. Howe e algina e/chi osan nanopa icles also appea as p omising
in o al deli e y sys em o insulin and po en ially o o he he apeu ical p o eins (107).
Mo eo e , i was demons a ed ha blood glucose le els o diabe ic a s can be e ec i ely
con olled by insulin-loaded chi osan nanopa icle adminis a ion, ollowing ei he single o
mul iple o al adminis a ion. In addi ion, he hypoglycemic e ec was obse ed o mo e
han 24 h (108).
2.2. Clinical ial - Sa e y and ole abili y o chi osa-n-ace ylcys eine eye
d ops in heal hy young olun ee s
The e a e many de ini ions o clinical ials, gene ally a e s udies o biomedical and heal h
esea ch ela ed o human beings ollowing a p e-de ined p o ocol (109). These es s can
only ake place when quali y and sa e y o he es a e gua an eed by he Heal h Au ho i y
o he E hics Commi ee ecognized by he coun y, which will un he clinical ial. The
andomized con olled ial is commonly accep ed as he gold s anda d esea ch me hod
o e alua ing heal h ca e in e en ions (110). In any clinical ial i is desi able no only o
achie e simila numbe s o pa ien s in each ea men g oup, bu also o ensu e ha
pa ien g oups a e simila wi h espec o p ognos ic ac o s such as age o s age o
disease (110). Nowadays, many s udies ha e come and explo e he mul i-po en ial o
chi osan as a ca ie o d ug deli e y and elease (109).
The "d y eye synd ome" DES is a highly p e alen ocula disease, pa icula ly in he
elde ly popula ion and ha cu en he apy is he use o opically adminis e ed lub ican s,
no "ideal" o mula ion has ye been ound. Recen ly, C oma Pha ma has in oduced
chi osan-n-ace ylcys eine eye d ops (111), designed o ea men o symp oms ela ed o
“d y eye synd ome". The new o mula ion comp ises n-ace ylcys eine, which has been
used in oph halmology because o i s mucoly ic p ope ies o se e al yea s. Based on
heo e ical conside a ions, one can hypo hesize ha he new chi osan de i a i e may
30
oxygen, en i onmen al chemicals and physical ab asion (137, 138). P o ec i e
componen s like wa e -soluble an ioxidan s, lipid-soluble an ioxidan s and highly
specialized enzymes a e hough o se e as a on line de ense o he ocula su ace ea
ilm and unde lying issues (118). I he an ioxidan s a e deple ed o ac ing ine icien ly, an
imbalance be ween he high inc eased p oduc ion o ROS and he sha p educ ion in
an ioxidan de enses will al e cellula edox s a us (136). In e inopa hy, OS has been
widely in ol ed in dec eased e inal blood low (139), inc eased ascula pe meabili y,
dis up ion o BRB (140) and he appea ance o cellula capilla ies om he apop o ic loss
o e inal capilla y cells (141). OS has also been linked o mic o ascula abno mali ies in
e inopa hies, degene a i e p ocess o e inal neo ascula iza ion and he supp ession o
an ioxidan s sys ems (25).
4. Nano echnology applied o an ioxidan s p o ec ion
Nanopa icles ha e been made o sa e ma e ials, including syn he ic bio-deg adable
polyme s, na u al biopolyme s, lipids and polysaccha ides and ha e he po en ial o
o e coming impo an mucosal ba ie s, such as ocula ba ie s (50). Nanopa icles may
be ob ained ia di e en p epa a ion p o ocols and ha e been widely s udied in ecen
yea s as ca ie s o he apeu ic agen s wi h a ying deg ee o e ec i eness (142).
Di e en p epa a ion o mic o/nanopa icles can be conside ed, mainly physical me hods,
chemical c osslinking me hods and miscellaneous. Physical me hodologies in ol e
iono opic gela ion, emulsi ica ion and iono opic gela ion, modi ied emulsi ica ion and
iono opic gela ion, loa ing hollow chi osan mic osphe es ob ained by ionic in e ac ion
wi h sodium dioc yl sul osuccina e, coace a ion and complex-coace a ion (34).
C osslinking wi h o he chemicals a e used o emulsion c osslinking me hod, mul iple
emulsion me hod, p ecipi a ion–chemical c osslinking and c osslinking wi h a na u ally
occu ing agen (34). Miscellaneous me hods include he mal c osslinking, sol en
e apo a ion me hod, sp ay d ying and in e acial acyla ion (34). Nanoca ie s can be
applied o imp o e he solubili y, pe meabili y, s abili y o he compounds and some can
e en su pass he i s pass me abolism (Figu e 1.4). These deli e y sys ems ha e been
bene icial o he pha maceu ical indus ies as i is a s a egic ool o expanding d ug
ma ke and pa en li e. No el d ug deli e y sys ems would make an ioxidan each si e o
ac ion and imp o e he e icacy o he apy, gene ally by imp o ing he bioa ailabili y,
which a e o p ime impo ance when an ioxidan s in ended o p ophylac ic pu pose (116).

31
Implica ion o no el deli e y sys ems o an ioxidan s is uled by physical-chemical
cha ac e is ics, biopha maceu ics and pha macokine ic pa ame e s o he an ioxidan o
be o mula ed (116). Recen ly, chemical modi ica ions, coupling agen s, liposomes,
mic opa icles, nanopa icles and gel-based sys ems ha e been explo ed o o e come
di icul ies in he de elopmen o new p oduc s o he imp o emen o human heal hca e
(18).
Figu e 1. 4. Classi ica ion o an ioxidan s and cha ac e iza ion o some nanos uc u es.
Highligh s o eal bene i s o an ioxidan s encapsula ion conside ing i s libe a ion,
abso p ion, dis ibu ion, me abolism, elimina ion and esponse, adap ed om (18).
Ne e heless, i may be highligh ed he huge impo ance o nano echnology and
nanoca ie s o be applied in common deli e y sys ems, like immunoglobulin’s deli e o
32
he e ina. In pa icula , he injec ing ascula endo helial g ow h ac o (VEGF) o
neu alize he an ibodies in o he i eous. Silica e nanopa icles (SiNPs) ha e been
demons a ed e iciency in inhibi ion o VEGF-induced angiogenesis. Via supp ession o
VEGF ecep o -2 phospho yla ion induced by VEGF, SiNPs blocked ERK 1/2 ac i a ion
(143). In a i eal injec ion o gold nanopa icle (GNP) also has shown an inhibi ion o
e inal neo ascula iza ion in a mouse model o e inopa hy o p ema u i y. GNP no
a ec ed he cellula iabili y o e inal mic o ascula endo helial cells and no induced
e inal oxici y. GNP can be used in a a iable aso-p oli e a i e e inopa hies media ed by
VEGF (144).
A molecule wi h ideal solubili y and pe meabili y p o ile can be adminis e ed wi h a
minimum e ec i e dose and wi h no p esys emic loss due o mucosas, physical ba ie s o
enzyma ic deg ada ion (18). Fo an ioxidan s assu e hese condi ions hey need
nanoca ie s o ake hem o he igh place wi hou losing hei unc ional ac i i y.
The e o e, conside ing he he apeu ic po en ial o he an ioxidan s, he e is e e y need o
implica e no el d ug deli e y echnologies o imp o e hei pe o mance. Nanopa icles
a e expec ed o de elop and imp o e p o ec ion, s abili y, bioa ailabili y and he e o e he
he apeu ic e icacy o an ioxidan s, wi hou comp omising he sa e y pe o mance o he
d ug. The Age-Rela ed Eye Disease S udy (AREDS) (145), an Na ional Eye Ins i u e–
sponso ed, mul icen e , con olled, andomized clinical ial, demons a ed ha he
combina ion o o al supplemen s consis ing o an ioxidan i amins C (500 mg), E (400
in e na ional uni s), and β-ca o ene (15 mg), and mine als, ZN (80 mg o zinc oxide) wi h
Cu (2 mg cup ic oxide). This educed he 5-yea isk o de eloping ad anced age macula
degene a ion (AMD) in eyes wi h in e media e AMD by 25% (es ima ed p obabili y o
p og ession was 28% o placebo s 20% o an ioxidan s plus zinc). The p ima y pu pose
o he Age-Rela ed Eye Disease S udy 2 (AREDS2) (145) was o e alua e he e ficacy
and sa e y o lu ein plus zeaxan hin and/o ω-3 long-chain polyunsa u a ed a y acid
(LCPUFA) supplemen a ion, educing he isk o de eloping ad anced AMD. Ne e heless
and besides he p omising e ec o his huge an ioxidan supplemen a y, i would be
expec able ha his e ec could be also po en ia ed by he p o ec ion o hese an ioxidan s
using he nano echnology. The p o ec ion may be c ucial in o de o gua an ee he sa e
an ioxidan pe o mance, bioa ailabili y and abso p ion, o e he se e al ba ie s o he
diges i e sys em (such as he biological luids and enzyma ic pa hways). O he s udies
ha e been pe o med o main ain o e en inc ease he ac i i y and s abili y o an ioxidan s
using nano echnology (Table 1.2). These a e e idences ha an ioxidan nanopa icles
33
may be used o he apy o se e al diseases, howe e u he s udies a e needed o p o e
hei e icacy in he p e en ion and ea men o e inopa hies.
Table 1. 2. Recen published da a o an ioxidan s nanoencapsula ion o se e al
applica ions.
An ioxidan
Me hod
Resul s
Applica ion
Re e ences
3,5-di- e -
bu yl-4-
hyd oxycinna
mic acid
A nanosilica-
immobilized
an ioxidan was
p epa ed and
inco po a ed in o
polyp opylene (PP)
by mel
compounding
The an ioxidan e iciency o he nanosilica-
immobilized an ioxidan was supe io o he
co esponding low molecula coun e pa
(AO). The he mal oxida i e s abili y o
PP/nanosilica-immobilized an ioxidan was
much highe han ha o PP/AO compound
du ing he long- e m accele a ed he mal
aging
Applica ion no
desc ibed
(146)
An ioxidan
enzymes
(ca alase and
supe oxide
dismu ase)
Magne ically
esponsi e
nanopa icles (MNP)
o med by
p ecipi a ion o
calcium olea e in he
p esence o
magne i e-based
e o luid (con olled
agg ega ion/
p ecipi a ion)
Ca alase s ably associa ed wi h MNP was
p o ec ed om p o eolysis and e ained 20%
o i s ini ial enzyma ic ac i i y a e 24 h o
exposu e o p onase. Unde magne ic
guidance ca alase-loaded MNP we e apidly
aken up by cul u ed endo helial cells
p o iding inc eased esis ance o OS (62 ±
12%) cells escued om hyd ogen
pe oxide induced cell dea h s. 10 ± 4%
unde non-magne ic condi ions
Can be highly
ele an o he
ea men o
ascula
disease
(147)
An ioxidan s o
Sal ia
mil io hiza
(sal ianolic
acid B,
c yp o anshino
ne, anshinone
I and
anshinone
IIA)
The d ied oo s
o S. mil io hiza
we e g ound by he
a omize and
u he sp ayed
g anula ing wi h he
aid o loa ing bed.
The esul ing
ma e ials we e d ied
o o m he
nanopa icles
unde he d y
p ocesses
S onge an ioxidan bioac i i ies we e
obse ed o he ex ac s p epa ed using
nano echnology in all es ed assays. The
pola ac i e cons i uen in he nanopa icles
samples was eleased as e compa ed o he
adi ionally powde ed samples
Applica ion no
desc ibed
(116)
Idebenone
Co-d ying wi h
chi osan (Ch) o N-
ca boxyme hylchi os
an (N-CMCh) c oss-
linked wi h
ipolyphospha e
(TPP)
Nanopa icles showed a 10- old inc ease o
d ug s abili y in compa ison wi h ee d ug
and p ese ed an ioxidan ac i i y in i o.
Compa ed wi h he se e ely i i a i e ee
o m o idebenone, he nanopa icle
o mula ion showed dec eased mucous
memb ane i i a ion
These
nanopa icles
ha e po en ial
oles a ca ie s
o hyd ophobic
and i i a i e
d ugs such as
he an ioxidan
idebenone o
opical o nasal
use
(148)
34
Na u al
an ioxidan s
ex ac ed om
Ilex
pa agua iensis
(ILE)
Nanopa icles we e
p epa ed by ionic
gela ion o chi osan
hyd ochlo ide and
sodium
ipolyphospha e.
The ac i e
componen s we e
added o he sodium
ipolyphospha e
solu ion and his was
added d opwise o
he chi osan
hyd ochlo ide
solu ion while s i ing
Chi osan hyd ochlo ide–TPP nanopa icles
main ain he an ioxidan ac i i y o ILE-
polyphenols. Nanopa icles eleased 100% o
ILE-polyphenols loaded a e 15 min in wo
bu e s wi h di e en pH alues (pH 5.7 and
6.5)
This me hod is
a p omising
echnique o
nu aceu ical
and cosme ic
applica ions
(149)
p-
Hyd oxybenzyl
alcohol (HBA)
HBA-inco po a ed
copolyoxala e
(HPOX)
HBA eleased om HPOX demons a ed
excellen an ioxidan ac i i y, such as
inhibi ion o ni ic oxide (NO) p oduc ion by
supp essing iNOS (inducible ni ic oxide
syn hases) exp ession in lipopolysaccha ide
(LPS)-ac i a ed RAW 264.7 cells. HPOX
nanopa icles deli e ed in anasally
signi ican ly educed pulmona y in lamma ion
and supp essed he iNOS exp ession
HPOX
nanopa icles
a e highly po en
o he ea men
o oxida i e
damage- ela ed
diseases, such
as as hma
(150)
Que ce in
Nanop ecipi a ion
echnique wi h
Eud agi ® E (EE)
and PVA as ca ie s
Pa icle dis ibu ion wi h polydispe si y index
<0.3, and i s yield and encapsula ion
e iciency we e o e 99%. The elease o he
d ug om he que ce in-loaded nanopa icles
(QUEN) was 74- old highe compa ed wi h
he pu e d ug. The an ioxidan ac i i y o he
QUEN was mo e e ec i e han pu e
que ce in on diphenyl-1-pic ylhyd azyl
(DPPH) sca enging, an i-supe oxide
o ma ion, supe oxide anion sca enging, and
an i-lipid pe oxida ion
These
nanopa icles
may be applied
in clinical se ing
(151)
Que ci in
(Albizia
chinensis
isola ed
an ioxidan )
Que ci in has been
encapsula ed on PLA
nanopa icles
by sol en
e apo a ion me hod
The encapsula ion e iciency o
nanoencapsula ed que ci in e alua ed by
HPLC and an ioxidan assay is 40%. The in
i o elease kine ics o que ci in unde
physiological condi ion e eals ini ial bu s
elease ollowed by sus ained elease. Less
luo escence quenching is obse ed wi h
equimola concen a ion o PLA encapsula ed
que ci in han ee que ci in. The p esence
o que ci in speci ic peaks on FTIR o i e
imes washed que ci in loaded PLA
nanopa icles p o ides an ex a e idence o
he encapsula ion o
que ci in in o PLA nanopa icles
Po en ial use o
he apeu ic o
in es inal an i-
in lamma o y
e ec and
nu aceu ical
compounds
(152)
α- ocophe ol
( i amin E)
Gold nanopa icles
p epa ed wi h
2,2-DPPH
Ch omanol g oups on gold nanopa icles
could e icien ly enhance he ac i i y o he
i amin E-de i ed an ioxidan
Po en ial
s a egy o
an ioxidan
design wi h
se e al
applica ions
(153)
35
4.1. Nanoan ioxidan s pha maco he apy
D ugs opically adminis e ed in he eye ha e low p obabili y o eaching he pos e io
segmen in signi ican amoun s, as hey ha e o pass h ough se e al me abolic and
physical ba ie s o each he e ina, namely he co neal and conjunc i al epi helium, hen
aqueous humo , and lens (132). One o he main p oblems encoun e ed wi h he opical
adminis a ion o liquid o ms is he apid and ex ensi e loss o d ugs o he limi ed
capaci y o d ug e en ion in he ocula su ace and also as a esul o he blinking
p ocess, which no mally is s imula ed a e ins illa ion (118). The BRB and he ex a ocula
epi helia ep esen he obs acle in he d ug deli e y o he cho oid, e ina and i eous.
Only a ac ion o he d ug adminis e ed o ally o by subcu aneous o in amuscula ou es
eaches he e ina, equi ing la ge doses o be he apeu ically e ec i e (118). P esen ly,
he e a e se e al an ioxidan p oduc s on ma ke which ha e been o mula ed in o hese
con en ional dosage o ms, wi h i amins leading he g oup. Vi amins ha e been
o mula ed mainly in o able s and capsules. Gene ally, hese agen s we e ound in
combina ions a he han indi idual p oduc s (18). A possible app oach o imp o e e inal
d ug deli e y is o acili a e localized deli e y o he pos e io segmen o he eye by using
Anopo e™ nanopo ous il e . Ca alase and i amin C we e deli e ed using hese ino ganic
nanopo ous il e , which is made up o aluminum oxide il e wi h po es o 20 nm size, as a
semipe meable memb ane o sepa a e wo compa men s in i o. The da a shown
ep esen ed he possibili y o biocompa ible capsules based on nanopo ous il e s, which
a e able o p o ide con olled deli e y o an ioxidan molecules (154). Se e al
nanoca ie s may be conside as nanoan ioxidan s anspo e s as sel -emulsi ying d ug
deli e y sys ems, which o e he po en ial o enhancing he abso p ion o poo ly soluble
and/o poo ly pe meable compounds. Fo d ugs ha a e poo ly soluble and/o poo ly
pe meable, a signi ican imp o emen in ep oducibili y in pe o mance and bioa ailabili y
migh be achie ed wi h hese nanoca ie s. Lu ein is a well-known an ioxidan and an i-
ee adical used in cosme ic, nu aceu ical indus y wi h po en ial applica ion in
pha maceu ics as suppo i e an ioxidan in ea men s. Howe e , lu ein is a lipophilic
molecule which is poo ly soluble in wa e and has a low bioa ailabili y. The lu ein
nanosuspension was con e ed in o pelle s and illed in o ha d gela in capsules o
nu aceu ical use, yielding a supe io in i o elease (155). Howe e , he e a e some
limi a ions associa ed wi h hese o mula ions, including s abili y, manu ac u ing me hods,
in e ac ion o he ill wi h he gela in shell and limi ed solubili y o some d ugs in lipid
sol en s (18). Liposomes a e po en ial sys ems o d ug deli e y because o hei size,

36
hyd ophilic and hyd ophobic cha ac e and biocompa ibili y (156). The e m liposomes
wi h an ioxidan s could be liposomes con aining lipid-soluble, wa e -soluble o enzyma ic
an ioxidan s. An ioxidan liposomes hold g ea p omise in he ea men o many diseases
in which OS plays a signi ican ole (157). A majo p oblem associa ed wi h con en ional
liposomes is ha hey a e ecognized by he immune sys em as o eign subs ances and
apidly emo ed by phagocy ic cells o he e iculoendo helial sys em (18). Nanopa icles
ha e been explo ed as d ug deli e y sys ems o encapsula ion o di e en d ugs o
inco po a ion ei he in o lipid o polyme ic pa icles. The nanoencapsula ion can help
deli e d ugs wi h poo aqueous solubili y and pe meabili y. In biodeg adable polyme ic
nanopa icles he d ug is dissol ed, adso bed, a ached o encapsula ed in he polyme ic
ma ix o nanome e size. Depending upon he me hod o p epa a ion, nanosphe es o
nanocapsules a e ob ained wi h di e en elease and su ace p ope ies (158).
Nanopa icles a e also being explo ed o a ge ed d ug deli e y (159). Polyme ic
nanoca ie s a e a ac i e ehicles o ascula d ug deli e y as well, bu emained a wai
echnology o an ioxidan enzymes due o poo loading and inac i a ion o p o eins du ing
o mula ion (159). Mic opa icles ha e also been designed and e alua ed as deli e y
sys ems o an ioxidan s p oduced endogenously and exogenously. The ma ix is
gene ally a polyme which sus ains he elease o d ug. These pa icles a e ei he ma ix
ype en apping he ac i e moie y o capsule ype encapsula ing he d ugs (18). The need
o imp o e he ocula d ug bioa ailabili y, e ec i eness and highe e en ion ime becomes
an eme ging ield in medicine, conside ing ocula condi ions. Al hough he e a e no any
an ioxidan nanopa icles agen app o ed by Eu opean Medicines Agency (EMA) and
Food and D ug Adminis a ion (FDA) he e a e a leas 60 agen s in he clinic ou ed as
an i-angiogenic, many mo e po en ial an i-angiogenic candida es a e cu en ly in
p eclinical de elopmen , wi h he dis inc possibili y o mo ing in o ocula clinical s udies
(160, 161). Recen da a om e y impo an mul icen e clinical ials ha e emphasized
he impo ance o new and e olu ional he apies.
4.2. Sa e y issues o an ioxidan nanopa icles
Despi e o nanopa icles wi h an ioxidan s a e known o be om na u al sou ces, hen
sa e, biocompa ible and biodeg adable wi h no oxici y e ec s o colla e al damages,
pha macokine ics and oxicological analyses should no be dispensable. This sa e y
37
pe o mance should gua an ee he molecula , subcellula and cellula beha io o
nanopa icles in ocula issues and in he sys emic ci cula ion. The de elopmen o in i o
cell cul u e models o s udying ocula ba ie s undoub edly p o ides a pla o m o
in es iga e he impac o pha maceu ical a icking on oph halmic diseases (162). The
human co neal epi helium HCE-T model, o med by ans ec ion o human co neal
epi helial cells om a 47-yea -old emale dono wi h a ecombinan SV40-adeno i us,
ep esen s a s anda d ool o d ug pe mea ion, bioa ailabili y p esc eening, and oxici y
assessmen (163), should be used o simula e he co neal abso p ion o an ioxidan s. The
cell line has good g ow h cha ac e is ics and shows a cobbles one-like appea ance. The
IOBA-NHC cell line, a non ans ec ed, spon aneously immo alized epi helial cell line
de i ed om human conjunc i a (164), should be used o p edic ing he conjunc i al
abso p ion o an ioxidan s. The IOBA-NHC cells demons a ed high p oli e a i e abili y in
i o and ypical epi helial mo phology. Fu he mo e, cy oke a ins, mannose, and sialic
acid esidues a e immunologically de ec ed, he e o e appea s ha his cell line can be a
use ul expe imen al ool in he ield o ocula su ace cell biology. Immo alized human
cells ARPE-19 cells a e pa icula ly impo an in he e inopa hies s udies, since hey
simula e he e inal beha io and may indica e he abso p ion o an ioxidan s. These cells
a e ully cha ac e ized ega ding hei mo phology, he exp ession o e ina speci ic
ma ke , and hei ba ie p ope ies (162, 165). Monolaye s o ARPE-19 cells ha e
become a well-es ablished in i o model o he ou e BRB. Mo eo e , monolaye s o
ARPE-19 cells a e used by esea che s o a a ie y o o he in i o expe imen s,
including s udies o he egula ion o gene exp ession, pola ized dis ibu ion and sec e ion
o p o eins, deli e y o genes and an isense oligonucleo ides, o oxici y s udies, and as
models o e inal diseases (162, 166, 167). The a e o nanopa icles loaded wi h na u al
an ioxidan s mus be ollowed wi h a minimum equi ed amoun o in i o expe imen s
using opical ou es o adminis a ion. The he apeu ic e icacy and sa e y in i o in abbi s
should be moni o ed by means o physiological and beha io al pa ame e s,
complemen ed by oxicological and moni o ing o conjunc i al issue.
38
5. Summa y
The physical-chemical p ope ies o chi osan, such as in e - and in amolecula hyd ogen
bonding and he ca ionic cha ge in acidic medium in addi ion wi h i s se e al bioac i i ies
such as non oxici y, biocompa ibili y, biodeg adabili y and ansmucosal abso p ion allows
chi osan o become an excellen candida e o d ug ca ie and excipien o con olled
elease sys ems. A numbe o chi osan-based colloidal sys ems ha e been explo ed o
bioac i e molecules ca ie s, like ilms, able s, hyd ogel, mic o and nanopa icle. In hese
o mula ions chi osan can each he a ge si es wi h low colla e al damages, oxici y and
highe e iciency. The an ioxidan he apy majo p oblem in ocula ea men s is he
di icul o main ain an e ec i e d ug concen a ion a he si e o ac ion o an app op ia e
pe iod o ime, in o de o achie e he expec ed pha macological esponse. This
subop imal deli e y can be o e come by di e en no el deli e y s a egies. Inno a i e
nanomedicines wi h an ioxidan s pha maco he apy could be seen as he key ac o o eye
diseases, since he use o opical nano-an ioxidan s o ea o delaying OS- ela ed ocula
mani es a ions is s ill unexplo ed, while cu en e inopa hies he apy includes in asi e
me hod like lase pho ocoagula ion o su ge y, which may also inc ease isk o
endoph halmi is, ca a ac o ma ion and e inal de achmen . Besides he de elopmen o
lase o e inopa hy, he e ha e been no majo ad ances in ea men o he disease,
despi e nume ous clinical ials. D ugs applied di ec ly o he eye ep esen a non-in asi e
and sa e me hodology, inc easing he e ec i eness o ea men and educing oxici y
associa ed wi h sys emic adminis a ion. An ideal an i-angiogenic agen should be
de eloped o neo ascula iza ion con ol and eg ession. I may inhibi and s abilize he
disease, o p e en he ision loss, e inal sca ing and de achmen wi h no oxici y as well
as he o mula ion should be o long e m d ug deli e y. Agen s should also be classi ied
in o ea ly and la e ac ing, speci ic and non-speci ic, and e e sible and i e e sible. The
unde s anding o whe e a d ug alls in o hese classes may help in he comp ehension o
he po en ial and/o limi a ion o he d ug when used in he clinic, as well as how o p edic
po en ial se ious ad e se e en s. The nano-an ioxidan s a e expec ed o be ea ly ac ing,
especially o p ophylaxis. Conside ing his, he e a e a numbe o challenges associa ed
o he ea men o ocula diseases. Thus, success ul al e na i es o ocula he apies a e
needed and hey should p o ide non-in asi e and a cos e ec i e ea men eaching
e e y economic s a us. The e o e, use ul knowledge o u u e adap a ion is p o ided o
pha maceu ical indus y, medical assis ance and human heal h ca e.
39
PART II - Aims and goals
“The e is a wo ld o ad an ages o nano echnology applica ion in di e en ields ...
a e all a pa o i s own disad an ages!”
Jacques de la Palice
46

47
Abs ac
In oduc ion: Sal ia o icinalis and Sa u eja mon ana (sage and sa o y, espec i ely) a e
plan s used in adi ional medicine. The quali y con ol o hei he bal o mula ions is o
pa amoun conce n o gua an ee he expec ed biological ac i i y o hei an ioxidan
compounds.
Objec i es: To es ablish a simple and e ec i e HPLC me hod o e alua e simul aneously
que ce in and osma inic acid, in a pu e o m, in na u al ex ac s (sage and sa o y), and
encapsula ed in o chi osan-based nanopa icles (in he nex sec ions).
Me hodology: Ch oma og aphy was pe o med on a RP C18 column, in a g adien mode
wi h a mobile phase comp ising me hanol: o mic acid:wa e 92.5:2.5:5 ( / ) a a low a e
o 0.75 mL/min and a de ec ion wa eleng h o 280 nm.
Resul s: The me hod was speci ic, linea in he ange o 0.05-1.0 mg/mL (R2 = 1.00),
p ecise a he in a-day and in e -day le els, accu a e ( eco e y a e pe cen age o 90.5 ±
0.6), and obus o changes in equipmen condi ions.
Conclusion: The es ablished me hod was e ec i e o que ce in and osma inic acid
cha ac e iza ion in na u al ex ac s and in chi osan nanopa icles, allowing he loading
capaci y de e mina ion, he associa ion e iciency as well as i s in i o elease.
48
49
CHAPTER 3 - High-pe o mance liquid
ch oma og aphy me hod alida ion
50
51
3. In oduc ion
Du ing he las decade, scien i ic e idences ha e p o ed ha plan phenolics a e an
impo an class o de ense an ioxidan s. These compounds a e i ually widesp ead in all
plan oods, o en a high le els, and include phenols, phenolic acids, la onoids, annins,
and lignans (6). Sage and sa o y a e plan s used in adi ional medicine, and g ow in he
poo soils o he Medi e anean basin (14). Besides applica ion as condimen s, sage and
sa o y ha e been used in olk medicine o hei an i-dia heal, diges i e, wound healing,
an i-in lamma o y, disin ec an , an i-hype ensi e and seda i e p ope ies. Some o hese
ac i i ies ha e been associa ed wi h hei high con en s o osma inic acid and he
p esence o o he ele an phenolic compounds such as, que ce in, u in and osma inic
acid (9, 14). Rosma inic acid (α-O-ca eoyl-3,4-dihyd oxyphenillac ic acid) is a phenolic
compound ha has been claimed o p o ide p o ec ion agains cance (7), among o he
biological ac i i ies, namely as ingen , an i-in lamma o y, an imu agen, an ibac e ial and
an i i al (7, 8). I is also an e icien na u al an ioxidan (9). Que ce in (3,3’,4’,5,7-
pen ahyd oxy la one) is a majo ep esen a i e o he la onol subclass (170). In i s
na u al sou ces i exis s mainly in he o m o glycosides and can be ound in ege ables,
ui s, he bs, o ed wine (151, 171). I has been demons a ed a a ie y o que ce in
biological ac i i ies and pha macological ac ions, such as dila ing co ona y a e ies,
dec easing blood lipid, an i-pla ele agg ega ion, an i-cance , an ioxidan , an i-anemia,
an i-in lamma ion, an i-anaphylaxis and hepa op o ec i e e ec s (151). Se e al s udies
ha e also epo ed ha que ce in can inhibi he p oli e a ion o mul iple cance cell ypes
(lung, colon, p os a e, panc ea ic ca cinoma cells) (171-173). Accu a e iden i ica ion and
quan i ica ion in inal o mula ions o plan ex ac s is essen ial o gua an ee he expec ed
biological ac i i y. The aim o his s udy was o alida e a HPLC me hod o simul aneous
quan i ica ion o osma inic acid and que ce in in na u al ex ac s and in polyme ic
nanopa icles o assess he con en , he associa ion e iciency, he in i o elease and
pe meabili y p o ile u he e alua ed in he nex chap e s.

52
3.1. Expe imen al
3.1.1. Ma e ials
Two plan s we e selec ed as ex ac sou ce namely sage and sa o y, bo h p o ided by
ERVITAL (Cas o Dai e, Po ugal), om a p e ious s udy in ol ing 48 medicinal plan s
made by ou esea ch g oup (115). These plan s had been cul i a ed as o ganic p oduc s,
and we e supplied in hei comme cial o m o d ied lea es: ca. 4 g was hen c ushed
(using a co ee mill) o 1 min, so as o ob ain he co esponding powde . A ac ion (ca. 1
g) was con ac ed, unde uni o m s i ing, wi h 100 mL o boiling dis illed wa e . A e he
powde deposi ion, samples we e il e ed, ozen a -80 ºC and lyophilized o u he
p ocedu es (He o Hol en A/S D ywinne ). Then solu ions (1%) o lyophilized powde we e
p epa ed in me hanol o ch oma og aphic analyses. Be o e injec ions, samples we e
il e ed h ough a 0.45 µm il e . Rosma inic acid (96.5 w/w, HPLC), que ce in dehyd a e
(99.0% w/w, HPLC), me hanol CHROMASOLV® (HPLC ≥ 99.9%) and o mic acid (HPLC
≥ 98.0%) we e pu chased om Sigma-Ald ich (Missou i, USA).
3.1.2. Equipmen and ch oma og aphic condi ions
All HPLC uns we e pe o med using a Wa e s Se ies 600 HPLC and esul s we e
acqui ed and p ocessed wi h Empowe ® So wa e 2002 o da a acquisi ion (Mild o d MA,
USA). HPLC analysis was conduc ed by using a No a-Pack® RP C18 column (250 x 4.6
mm i.d., 5 µm pa icle size and 125 Å po e size) om Wa e s. Ch oma og aphic analysis
was pe o med in g adien mode. The mobile phase consis ed o me hanol: o mic
acid:ul a-pu e wa e in he a io o 92.5:2.5:5 ( / ). S a iona y phase was made wi h he
same componen s in he a io o 5:2.5:92.5, espec i ely. The phases we e il e ed
h ough 0.22 µm il e and degassed. Eluen was pumped a a low a e o 0.75 mL/min,
he injec ion olume was 20 μL and de ec ion wa eleng h was se o 280 nm. All
expe imen s occu ed a oom empe a u e and he o al a ea o peak was used o
quan i y he osma inic acid and que ce in compounds. The condi ions we e in es iga ed
o p o ide a simple p ocedu e wi h he bes peak esolu ion ega ding symme y and
ailing, educed un ime and cos -e ec i e analysis.
53
3.1.3. P epa a ion o s anda d and sample solu ions
S ock s anda d solu ions o 2 mg/mL o osma inic acid and que ce in we e p epa ed wi h
me hanol. The calib a ion cu e was made om he dilu ion o s ock solu ions in me hanol
o se en s anda ds: 0.05, 0.1, 0.2, 0.3, 0.5, 0.8 and 1.0 mg/mL.
3.1.4. Me hod alida ion
The HPLC me hod was alida ed acco ding o he In e na ional Con e ence on
Ha moniza ion (ICH) guidelines (174), using he ollowing analy ical pa ame e s: linea i y,
p ecision, accu acy, speci ici y, ange, obus ness, de ec ion and quan i ica ion limi s.
Linea i y was e alua ed by calcula ion o a eg ession line using leas squa es me hod.
Calib a ion cu es we e ob ained om se en di e en concen a ions analyzed h ee
imes. P ecision was assessed by es ing he epea abili y o h ee di e en s anda d
solu ions en imes (in a-day) and by in e media e p ecision analyzing he same h ee
s anda d solu ions, h ee imes on di e en days (in e -day). Accu acy was es ed by
pe cen age eco e y o mean o h ee de e mina ions o osma inic acid and que ce in a
h ee di e en concen a ions p ecisely p epa ed and by de e mina ion o he ela i e
s anda d de ia ion (RSD). Speci ici y was de e mined by compa ing osma inic acid and
que ce in samples unde di e en s ess condi ions ha may common a ec na u al
ex ac s and an ioxidan ac i i y as he empe a u es o (60, 20 and 1 ºC) o 24 and 72 h.
The solu ions we e also subjec ed o he e ec o ligh and ai in he same pe iod o ime.
Range was de i ed om linea i y, accu acy, and p ecision s udies. Robus ness was
e alua ed by es ing he same ch oma og aphy condi ions in di e en HPLC equipmen
(Me ck-Hi achi In e ace D-7000). De ec ion limi (DL) and quan i ica ion limi (QL) we e
de e mined based on he s anda d de ia ion o he esponse and on he slope o he
calib a ion cu e, using he ollowing exp essions:
𝐷𝐿= 3.3𝜎
𝑆
𝑄𝐿=10𝜎
𝑆
54
whe e σ is he s anda d de ia ion o he esponse and S is he slope o he calib a ion
cu e.
3.1.5. Me hod applicabili y
Applicabili y o he me hod was go e ned wi h he simul aneous de e mina ion o wo
an ioxidan compounds wi h g ea impac on heal h. The de e mina ion could be made o
each independen compound o combined, since he p esence o bo h does no a ec
hei quan i ica ion, also i could be made in na u al ex ac s o e en in he nanopa icula e
sys ems. This HPLC me hod was also de eloped, op imized and alida ed o he bes
p ecise esul s ega ding, osma inic acid associa ion e iciency in chi osan-based
nanopa icles, osma inic acid in i o elease om he a o men ioned nanopa icles and i
pe meabili y p o ile in cell monolaye s, assays de eloped and cha ac e ized in he nex
chap e s.
3.2. Resul s and discussion
3.2.1. Applica ion o he ch oma og aphic me hod
An HPLC me hod o he assessmen o osma inic acid and que ce in has been
p oposed. P e ious expe iences we e exploi ed o p o ide a simple p ocedu e wi h he
bes ch oma og aphic peak esolu ion, educed un ime and cos -e ec i e analysis. All
hese ac o s con ibu e o he es ablishmen o an analy ical me hod which pe mi s he
analysis o a la ge se ies o samples. A ypical ch oma og am o he p oposed me hod
was depic ed in Figu e 3.1. The osma inic acid and que ce in peak e en ion ime was
48.9 ± 0.1 min (i) and 57.9 ± 0.6 min (ii), espec i ely. These e en ion imes we e no
comple ely s aigh , bu his me hod may also be use ul o he iden i ica ion and de ec ion
o o he compounds in na u al ex ac s, which we e complexes ma ixes wi h di e en
compounds (la es e en ion ime compound a 90 min). Howe e , e en ion ac o
pa ame e (k’) was measu ed in o de o e alua e he ch oma og aphic pe o mance
55
(175). This e en ion ac o was o en used o desc ibe he mig a ion a e o an analy e on
a column. The e en ion ac o o analy e A was de ined as:
𝐾′𝐴=𝑡𝑅−𝑡𝑀
𝑡𝑀
whe e e en ion ime o he analy e ( R) and e en ion ime o he eluen ( M) we e easily
ob ained om he ch oma og am. When he analy ic e en ion ac o was less han one,
elu ion was so as ha accu a e de e mina ion o he e en ion ime was e y di icul .
High e en ion ac o s (g ea e han wen y) mean ha elu ion akes a e y long ime.
Ideally, he e en ion ac o o an analy e was be ween wo and en (175). The e en ion
ac o s o bo h compounds we e 0.918 and 0.930, o osma inic acid and que ce in,
espec i ely. Bo h compound pa ame e s we e in he op imal ange (k’ < 10), which
means ha he mig a ion a e o he analy es we e adequa e and so was he me hod. A
good expe imen al design is c ucial, and his seems pa icula impo an in he bal
medicines in o de o ob ain an op imal sepa a ion, since hese na u al ma ixes may
en ail hund eds o na u al compounds (176).
3.2.2. Linea i y
Linea i y was s udied in he concen a ion ange o 0.05-1.0 mg/mL by isual inspec ion o
a calib a ion cu e plo ing (n = 21) and by calcula ing he eg ession equa ion and he
co ela ion coe icien (R2) by he me hod o leas squa es. A p ima y s ock solu ion was
accu a ely p epa ed ollowed by igo ous dilu ion o gi e seconda y s anda d solu ions.
Each sample was analyzed h ee imes. A good linea i y was ob ained in he ange o
s udy. The calib a ion cu e o osma inic acid was:
𝐴=5.44 × 107 (±2.66 × 105 ) × 𝐶− 6.21×105 (±1.43 × 105)(𝑛=21) 𝑅2=1.00
and he calib a ion cu e o que ce in was:
𝐴=4.48 × 107 (±2.73 × 105 ) × 𝐶− 9.75×105 (±1.47 × 105)(𝑛=21) 𝑅2=1.00
whe e A is he peak a ea, C is he s anda d solu ion concen a ion in mg/mL, and
s anda d de ia ion alues o A and C a e indica ed in b acke s. The R2 ob ained was
highe han 0.999 as equen ly ecommended (177), indica ing a good linea i y in he
p oposed ange.
62

63
PART IV
“Science is a way o hinking much mo e han i ’s a body o knowledge”
Ca l Sagan
64
65
Abs ac
In oduc ion: Nano echnology can be applied o deli e and p o ec an ioxidan s in o de o
con ol he OS phenomena in se e al ch onic pa hologies. Chi osan nanopa icles a e
biodeg adable ca ie s ha may p o ec an ioxidan s wi h po en biological ac i i y such as
osma inic acid in Sal ia o icinalis (sage) and Sa u eja mon ana (sa o y) ex ac s o sa e
and inno a i e he apies.
Objec i e: De elopmen , op imiza ion and cha ac e iza ion o chi osan-based
nanopa icles as s able and p o ec i e ehicle o deli e osma inic acid o medical
applica ions using na u al ex ac s as sage and sa o y.
Me hodology: An ioxidan -chi osan based nanopa icles we e p epa ed by ionic gela ion
wi h sodium ipolyphospha e (TPP), a pH 5.8 wi h mass a io o 7:1 (chi osan:TPP), wi h
a heo e ical an ioxidan -chi osan loading o 40 o 50%. The size and shape o
nanopa icles we e hen cha ac e ized by di e en me hods such as: pho on co ela ion
spec oscopy, lase Dopple anemome y, scanning elec on mic oscopy (SEM) and
ansmission elec on mic oscopy (TEM). Chemical in e ac ions be ween an ioxidan s and
chi osan we e assessed by di e en ial scanning calo ime y (DSC) and Fou ie - ans o m
in a ed (FTIR). HPLC allowed he associa ion e iciency and in i o eleased
measu emen s. An ioxidan ac i i y was e alua ed by 2,2-azinobis (3-
e hylbenzo hiazoline-6-sulphonic) (ABTS) and oxygen adical abso bance capaci y
(ORAC) me hods be o e and a e lyophiliza ion o assu e ha an ioxidan ac i i y was no
comp omised a e he d ied p ocess.
Resul s: Small sizing nanopa icles, a ound 300 nm, we e ob ained. SEM and TEM
con i med smoo h and sphe ical nanopa icles. No chemical in e ac ions we e ound
be ween an ioxidan s and chi osan, a e encapsula ion, by DSC and FTIR. The
associa ion e iciency was 51.2% o osma inic acid (wi h 40% loading), 96.1 and 98.2%,
o sage and sa o y nanopa icles, espec i ely (bo h wi h 50% loading). The bes
an ioxidan ac i i y esul s we e ob ained a e nanopa icles lyophiliza ion and by ORAC
me hod, he alues o osma inic acid, sage and sa o y nanopa icles we e: 3.6520 ±
0.1770, 0.4251 ± 0.0069 and 0.4526 ± 0.0087 µmol/eq T olox, espec i ely.
Conclusion: The ex ac s unde s udy we e p omising ehicles o osma inic acid d ug
deli e y in chi osan nanoca ie s.
66
67
CHAPTER 4 - De elopmen , op imiza ion and
physical-chemical cha ac e iza ion o chi osan-
based nanopa icles

68
69
4. In oduc ion
Sage and sa o y, a e plan s o en used in adi ional medicine o imp o e diges ion (179),
as disin ec an (180), o dec ease blood p essu e (7), o p e en p ema u e ejacula ion
(181), o ea neu opa hy (182), u ina y and pulmona y in ec ions (183), and o he
diseases such as Alzheime ’s disease (184) and cance (183). Some o hese biological
ac i i ies ha e been associa ed wi h i s high con en s in osma inic acid (9, 179, 185).
Rosma inic acid, (a-O-ca eoyl-3,4-dihyd oxyphenillac ic acid), is a phenolic compound
gene ally admi ed as a ee adical sca enge (125). Besides i s huge an ioxidan ac i i y
osma inic acid can ha e many bene icial unc ionali ies like an ibac e ial and an i i al
ac i i y, an i-in lamma o y ac i i y (127), an i-mu agenici y cha ac e (128), capabili y o
educe a opic de ma i is symp oms (129), p e en ion o Alzheime ’s disease (130) and
apop osis induc ion o colo ec al cance cells (131). Ne e heless, besides he poo
abso p ion ha cons ain he anspo ac oss biological ba ie s, mos o he na u al
an ioxidan s o o he ac i e compounds a e uns able and mus be p o ec ed om
deg ada ion in he physiological en i onmen (186). Thus, he e icacy o hese d ugs
clea ly depends on he design o app op ia e ca ie s o hei deli e y, p o ec ion and
elease (1). Among he di e en app oaches explo ed so a , colloidal ca ie s ha e
pa icula in e es , especially hose made o mucoadhesi e polyme s o assu e d ug ime
e en ion a he abso p ion si e (2). Fo his applica ion, chi osan has become o
pa icula ly in e es ing o he associa ion and deli e y o labile mac omolecula
compounds, due o i s excep ional po en ial o d ug deli e y, especially o mucosal,
con ol d ug elease and p o ec agains ad e se condi ions like mucosal enzymes and
biological p o ec i e luids (187). The sma symbiosis o hese chi osan nanopa icles wi h
a high po en an ioxidan could be a hope o u u e he apies, conside ing he impo an
e ec o OS in se e al ch onic pa hologies. In his s udy chi osan nanopa icles we e
used, o inco po a e na u al ex ac s o sage and sa o y as a s able and p o ec i e ehicle
o deli e osma inic acid o medical applica ions. The e we e no epo s in he li e a u e
ha ha e demons a ed he good pe o mance o chi osan nanopa icles o inco po a e
hese ex ac s in o de o b ing he huge bene i s o osma inic acid an ioxidan , as well as,
o he compounds ha may ac syne gis ically.
70
4.1. Expe imen al
4.1.1. Ma e ials
The wo selec ed plan s sage and sa o y we e p o ided by ERVITAL (Cas o Dai e,
Po ugal). The plan s had been cul i a ed as o ganic p oduc s, and we e supplied in hei
comme cial o m o d ied lea es. The d ied lea es we e hen kep in he da k a 20 ºC.
Rosma inic acid (pu i y 96.5%), me hanol CHROMASOLV® (HPLC ≥ 99.9%) and o mic
acid (HPLC ≥ 98.0%) we e pu chased om Sigma-Ald ich (Missou i, USA). Chi osan low
molecula weigh and sodium ipolyphospha e (TPP) we e also pu chased om Sigma-
Ald ich (Lisbon, Po ugal). The deg ee o deace yla ion o he low molecula weigh
(LMW) chi osan was 85%, wi h a pu i y g ade o 85%. Pu e ace ic acid was pu chased
om Po nalab (Lisbon, Po ugal). Sodium hyd oxide (NaOH) and hyd ochlo ic acid (HCl)
we e om Me ck (Da ms ad , Ge many). Ul a-pu e wa e was ob ained in he labo a o y
using a Millipo eTM wa e pu i ica ion equipmen (Massachuse s, USA).
4.1.2. P epa a ion o chi osan-based nanopa icles
Op imized condi ions o ob ain chi osan nanopa icles we e based on p e iously s udies
(188), and was schema ically desc ibed in Figu e 4.1. The chi osan nanopa icles we e
ob ained by inducing he gela ion o a chi osan solu ion wi h TPP. Chi osan was dissol ed
in ace ic acid aqueous solu ions a a ious chi osan concen a ions: 0.05, 0.5, 1, 2, 3 and
5% (w/ ), he pH alue was adjus ed o 5.8 wi h 1M NaOH. The concen a ion o ace ic
acid was, in all cases, 1.75 highe han chi osan. Then, TPP was dissol ed in pu i ied
wa e a 0.05, 0.1, 0.2, 0.5, 1 and 2% (w/ ). Fo he s udy o he bes a io chi osan:TPP, a
olume o he TPP solu ion o 2 was added o 5 mL o he chi osan solu ion unde
magne ic s i ing a oom empe a u e, hus achie ing a inal concen a ion o 2 mg/mL
and 0.28 mg/mL o chi osan and TPP espec i ely (7:1). S ock solu ion o chi osan (1%)
and TPP (0.1%) we e main ained a 4 ºC o a pe iod o 1 mon h.
71
Figu e 4. 1. Schema ic illus a ion o ionic gela ion p ocess o an ioxidan -chi osan based
nanopa icles.
4.1.3. Encapsula ion o sage, sa o y and osma inic acid in o chi osan-based
nanopa icles
The addi ion o 1% ex ac s aqueous solu ion and a 1% o aqueous osma inic acid
solu ion was added o chi osan p e iously dissol ed in ace ic acid a a pH alue adjus ed
o 5.8, in di e en olumes in o de o gua an ee he bes concen a ion a io be ween
chi osan and he di e en compounds. The encapsula ion o osma inic acid, sage and
sa o y we e es ed in di e en heo e ical loadings (5, 10, 15, 20, 30, 40 and 50%) ai ly o
he ini ial concen a ion o chi osan (2 mg/mL). All he es s we e made o he 7 ba ches,
conside ing he wo plan s and he an ioxidan pu e. Some o he inal ba ches we e hen
lyophilized (He o Hol en A/S D ywinne ) and main ained a -20 ºC o 1-2 mon hs o
u he analysis.
4.1.4. Size and su ace cha ge
Size and polydispe si y (size dis ibu ion) o eshly loaded nanopa icles we e de e mined
by pho on co ela ion spec oscopy using Ze aPALS (B ookhea en, New Yo k, USA). A
sample o 1.6 mL was gen ly homogenized, placed in o analyze chambe and measu ed.
Collec i e 6 eadings we e pe o med h ee imes on a sample o pa icles a 25 ºC wi h a
de ec ion angle o 90º. The ze a po en ial was de e mined by lase Dopple anemome y,
Rosma inic
acid/
Sage/Sa o y
TPP
Chi osan
An ioxidan -
chi osan based
nanopa icles
s i ing
78
main ained a e eeze d ied (dehyd a ion p ocess). In he SEM analysis he pa icles
a e lyophiliza ion also con i med smoo h and sphe ical shape wi h size below 500 nm,
e en wi h some agg ega ion due o he d y p ocess (196). I can be he e o e a i med ha
osma inic acid and ex ac s encapsula ion (ei he esh o lyophilized), did no
conside ably a ec pa icle shape and o e all size as i was desc ibed abo e. Figu e 4.3
shows he SEM images o lyophilized chi osan nanopa icles p epa ed by ionic gela ion
unde he same pH condi ions, o he encapsula ion o osma inic acid in a pu e o m,
sage and sa o y. The lyophilized samples e en wi h some agg ega ion o he
nanopa icles o med by dispe sion du ing eeze d ying gua an ee he nanoscale pa icles
wi h he diame e s, which was in acco dance wi h o he p e ious s udies (197). The
mic os uc u al analysis con i med he mo phology and size o he nanopa icles. O he
s udies epo ed an inc ease in he pa icle size a e lyophiliza ion wi h he unmodi ied
chi osan pa icles (198). This was esul ed om agg ega ion om he s ong in e - and
in a-molecula hyd ogen bonding, which was no possible o b eakdown e en by o ex
homogeniza ion (198). This pa icles size inc ease a e lyophiliza ion p ocess was also
epo ed in essen ials oils encapsula ion (197).
Figu e 4. 3. SEM mic og aphs o lyophilized chi osan-based nanopa icles loaded: (a).
comme cial osma inic acid; (b). sage; and (c). sa o y.
1µm
1µm
1µm
1µm
1µm
1µm
(a)
(b)
(c)

79
4.2.3. Associa ion e iciency and d ug loading
I is known ha chi osan in acidic media (pKa 6.5) can in e ac wi h he nega i ely cha ged
TPP, o ming in e - and in a-molecula c oss-linkages, yielding ionically c osslinked
chi osan nanopa icles (36). This is a spon aneous me hod o smalle nanopa icles
o ma ion wi h posi i e cha ge, wi hou using any o ganic sol en o su ac an s (198). I is
also known ha he in e - and in a-molecula linkages c ea ed be ween TPP and he
posi i ely cha ged amino g oups o chi osan a e esponsible o he success o he
gela ion p ocess (188). In he p esen s udy i was desc ibed a nanopa icula e sys em
able o encapsula e na u al ex ac s. The pa icle size was obse ed o be dependen on
bo h chi osan and TPP concen a ions as desc ibed in p e iously s udies (188), being he
minimum sizes ob ained o he lowes chi osan and TPP concen a ions. Fu he
expe imen s we e conduc ed using he mass a io chi osan:TPP o (7:1), whe e TPP inal
concen a ion was 0.28 mg/mL and chi osan inal concen a ion was 2 mg/mL. Rosma inic
acid was selec ed as a model an ioxidan in o de o in es iga e he easibili y o using
chi osan and chi osan nanopa icles o na u al ex ac ca ie s. Associa ion e iciency and
heo e ical an ioxidan loading o hese nanopa icles we e displayed in Table 4.2.
The pH o he nanopa icles o ma ion medium was be ween 5.8 and 6.0, a pH alue ha
a o s he in e ac ion o osma inic acid and chi osan, hus eaching a maximum leading
o he en apmen o high amoun s o osma inic acid. Among he di e en samples
conside ing he encapsula ion o osma inic acid in o chi osan nanopa icles, i was
obse ed simila associa ion e iciency a ound 50% (Table 4.2), o all he di e en
loadings no signi ican di e ences we e obse ed (P > 0.05). Highe associa ion e iciency
was ound o osma inic acid en apmen in ex ac s o sage and sa o y, 96 and 98%
espec i ely wi h no signi ican di e ences obse ed (P > 0.05). This was in acco dance
wi h p e ious epo s (190).The highe associa ion e iciency in ex ac s nanopa icles may
be due o he di e en amoun o osma inic acid in chi osan nanopa icles and inside he
ex ac s in chi osan nanopa icles (Table 4.2). Since compe i i e in e ac ion may be
happen be ween phenolic (OH−) o osma inic acid and (P3O105−) g oups o TPP o
p o ona ed amino g oups o chi osan esul ing in low le els o pa icle o ma ion compa ed
o he chi osan nanopa icles, his may be in ensi ied wi h he highes amoun o
osma inic acid and phenolic g oups (198). This was in acco dance wi h o he s udies ha
encapsula e o he phenolic compound, such as ca hechin, in chi osan nanopa icles (198)
and in o he nanopa icles (199).
80
Table 4. 2. Associa ion e iciency, heo e ical loading, and inal osma inic acid con en in
chi osan nanopa icles.
Nano
AE
(%)
Theo e ical
Loading
(%)
Final con en
in he chi osan
nanopa icles
(µg/mL)
Final osma inic
acid con en
in chi osan
nanopa icles
(mg/mL)
Rosma inic acid
51.2 ± 3.0
40
800
400
Sage
96.1 ± 0.2
50
1000
100
Sa o y
98.2 ± 0.1
50
1000
50
The associa ion e iciency alues we e highe han he esul s o Ilex pa agua iensis
en apmen in calcium algina e nanopa icles coa ed wi h chi osan, (a ound 50%) since
ac i e compound was los du ing imme sion in chi osan (200). The good esul s o sage
and sa o y associa ion e iciency may be due o he huge a ini y o chi osan and his wo
c ude ex ac s. The esul s we e also highe han a s udy epo ed o que ci in
encapsula ion in o nanopa icles, which was only 40% (152). Howe e he high
associa ion e iciency epo ed in his s udy, was in acco dance o o he p e iously s udies
wi h he encapsula ion o na u al an ioxidan s ex ac ed om Ilex pa agua iensis in o
chi osan nanopa icles (193) and o he encapsula ion an ioxidan idebenone-loaded in o
chi osan nanopa icles (201).
4.2.4. In i o osma inic acid elease om chi osan nanopa icles
All he chi osan-based nanopa icles suspension and he PBS used as medium elease
we e adjus ed o he ea no mal osmola i y (300 mOsm / L). This no mal osmola i y is
essen ial o main ain cellula olume, enzyma ic ac i i y, and cellula homeos asis (202).
In his in i o elease s udy e e y e o was made in o de o bes a e he ocula
physiological condi ions o es he kine ic nanopa icles beha io in ocula su ace. Du ing
he expe imen a as elease was obse ed du ing he in i o elease assay which was in
acco dance wi h p e ious da a (190). The di e en osma inic acid con en s (in osma inic
acid nanopa icles, sage and sa o y nanopa icles) we e eleased in all he o mula ions
eshly and lyophilized wi hin 60 min wi h no signi ican di e ences (P > 0.05). An ini ial
bu s e ec was obse ed in he i s 30 min wi h app oxima ely 80% in sage nanopa icles
and almos 100% in osma inic acid and sa o y nanopa icles (Figu e 4.4). The esul s
81
seem o demons a e ha a signi ican amoun o osma inic acid o ex ac s we e ini ially
associa ed wi h nanopa icles on hei su aces by weak linkages o chi osan, which did
no ha e he necessa y s eng h o en ap all he compounds. This ep esen s ha
chi osan nanopa icula e sys em could e ain he p ima y s uc u e o osma inic acid o
ex ac s du ing encapsula ion. The p o ec ion elease happens only o ew minu es due o
he polyme ne wo k, which was in acco dance wi h o he s udies, ha encapsula e
polyphenols om Ilex pa agua iensis in chi osan nanopa icles p epa ed also by ionic
gela ion and a comple e elease o 100% we e demons a ed in he i s 15 min (193). In
ano he s udy o que ce in encapsula ion in o o he nanoca ie s, he elease wi hin he
i s 20 min was also 95% (151). Ne e heless, his esul s we e di e en om o he wo ks
ha demons a ed a comple e elease o polyphenols om chi osan nanopa icles wi hin 4
h (198). The e icien applica ion o hese nanoca ie s will ce ainly depend o he d ug
pu pose. Chi osan nanopa icles main ain hei cha ac e is ics o his heo e ical loadings,
and his con e s hem aluable p ope ies, such as p o ec i e and mois u ize , o he
encapsula ion o ac i e agen s o cosme ic o ocula applica ions, since a apid eleased
is in en ional (193). Howe e o o he applica ions, like o al d ug deli e y a slowe elease
should be op imized.
Figu e 4. 4. Rosma inic acid in i o elease om osma inic acid, sage and sa o y
chi osan-based nanopa icles.
0
20
40
60
80
100
120
020 40 60 80 100 120
Rosma inic acid (%)
Time (min)
Rosma inic acid nanopa icles Sage nanopa icles
Sa o y nanopa icles
82
4.2.5. The mal beha io by di e en ial scanning calo ime y analysis
The DSC measu emen s p o ide quan i a i e and quali a i e in o ma ion abou physical
and chemical changes ha in ol e endo he mic o exo he mic p ocesses, o changes in
hea capaci y. Endo he mic and exo he mic peaks co espond o ansi ions ha abso bs
o elease ene gy, espec i ely. DSC was pe o med o unde s and he beha io o
osma inic acid, sage and sa o y loaded and unloaded chi osan nanopa icles and he
he mog ams we e displayed in Table 4.3.
Table 4. 3. Peak empe a u es in he DSC he mog ams collec ed om chi osan,
osma inic acid, sage and sa o y, physical mix u es, and nanopa icles.
T (oC)
Onse
Peak
EndSe
Nano
Chi osan
102 ± 0.55
122 ± 1.20
131 ± 0.12
Rosma inic acid
112 ± 0.61
130 ± 0.73
141 ± 0.24
Sage
83 ± 0.27
108 ± 0.71
118 ± 0.93
Sa o y
103 ± 1.11
124 ± 0.82
137 ± 0.44
F ee
Rosma inic acid
134 ± 0.13
144 ± 0.23
149 ± 0.25
Sage
140 ± 0.21
141 ± 0.14
145 ± 1.03
Sa o y
141 ± 0.53
154 ± 0.51
158 ± 0.81
Physical mix u e
Chi osan-Rosma inic acid
90 ± 0.38
122 ± 0.83
136 ± 0.14
147 ± 0.14
150 ± 0.70
154 ± 0.13
Chi osan-Sage
94 ± 0.91
121 ± 0.10
134 ± 0.50
147 ± 0.67
151 ± 0.63
155 ± 0.84
Chi osan-Sa o y
93 ± 0.70
121 ± 0.32
132 ± 0.32
145 ± 0.28
151 ± 0.64
154 ± 0.24
No e: The esul s we e gi en as mean o iplica e samples.
The same chi osan-based nanopa icles he mal beha io was obse ed in all
he mog ams (Figu e 4.5Ia, IIa and IIIa). In all chi osan cu es, an endo he mic peak nea
70°C can be asc ibed o he loss o wa e as p e iously epo ed (203). The endo he m o
osma inic acid, sage and sa o y nanopa icles showed di e en shi empe a u es
(Figu e 4.5Id. IId and IIId, espec i ely). This may be accoun ed by he hyd ophilic g oups
83
inco po a ed due o osma inic acid ha we e in di e en amoun s inside he ex ac s
pa icles. O he p e iously s udies also epo ed simila shi s in DSC plo s o chi osan and
chi osan nanopa icles (198, 204). I could also be seen ha he peaks o he complexes
we e shi ed om hose o physical mix u e. Peaks o physical mix u e (Figu e 4.5Ic,
4.5IIc, 4.5IIIc) appea ed o be combina ions o each ma e ial bu hey we e di e en om
hose o nanopa icles, p obably because complexa ion o polyelec oly es, in acco dance
wi h o he simila wo ks (205). Also, compa ing endo he mic peak o an ioxidan s loaded
chi osan nanopa icles o he one ob ained wi h unloaded nanopa icles, he o me s a ed
a highe empe a u e, which was a possible e idence o he p esence o an ioxidan s
once i s decomposi ion s a ed a highe empe a u e when compa ing o unloaded
nanopa icles. The osma inic acid, sage and sa o y loaded sample showed he simila
shi o chi osan nanopa icles, which con i ms ha he e we e no signi ican co alen
in e ac ions be ween an ioxidan s and chi osan a e encapsula ion and c oss-linking
(205).

84
Figu e 4. 5. The mog am o : I.(a). chi osan emp y nanopa icles; (b). ee osma inic acid;
(c). osma inic acid and chi osan physical mix u e (mixing a io 1:1); (d). osma inic acid
encapsula ed in chi osan nanopa icles (a a heo e ical 40% loading) (d). II.(a). chi osan
85
emp y nanopa icles; (b). ee sage; (c). sage and chi osan physical mix u e (mixing a io
1:1); (d). sage encapsula ed in chi osan nanopa icles (a a heo e ical 50% loading).
III.(a). chi osan emp y nanopa icles; (b). ee sa o y; (c). sa o y and chi osan physical
mix u e (mixing a io 1:1); (d). sa o y encapsula ed in chi osan nanopa icles (a a
heo e ical 50% loading).
4.2.6. Spec oscopy by Fou ie - ans o m in a ed analysis
S uc u al ea u es, unc ional g oups ha ep esen backbone p oduce cha ac e is ic and
ep oducible abso p ions in he spec um, which can be analyzed by FTIR. Wi h hese
se ies o expe imen s i was in ended o moni o ize he complexa ion o con a y cha ged
polyelec oly es a speci ic pH and s oichiome ic ela ionship be ween he polyelec oly es
and an ioxidan s in nanoca ie s. Fo his conce n and o examine his ela ionship
be ween componen s o nanopa icula e sys ems, p elimina y conce ns we e aken o e
polyelec oly es in e ac ions and an ioxidan s en apmen . I is well es ablished ha he
ca boxyl g oup (–COO) o he anionic polyme may in e ac wi h he amino g oup ð-NH3
o chi osan and o m an ionic complex be ween he wo compounds (205). Rosma inic
acid displays a ypical ib a ional abso p ion bands wi h he main bands loca ed be ween
1800 and 700 cm–1 (206). The h ee bands a ound 1605, 1520, and 1445 cm–1 we e due
o he p esence o a oma ic ings in he molecule indica ing an a oma ic ing s e ching
(206). O he e idences o phenolic g oups we e deli e ed h ough he bands a 1360 and
1180 cm–1 esul ing om O-H and C-O s e ches (206). The e o e, he band a 1684 cm–1
and he wo shoulde s ecognized wi h his band esul p obably om he shi ed bands
due o he p esence o ca boxylic acid g oups and es e g oup 1725-1750 cm–1 (207).
Figu e 4.6 (I, II and III) showed ha all he abo e cha ac e is ic peaks appea in he
spec a o combined d ugs loaded chi osan nanopa icles a he same wa enumbe
indica ing no modi ica ion o in e ac ion be ween he d ug and ca ie . This was in
acco dance o p e ious wo k (208). Ne e heless, some peaks clea ly dec eased in
in ensi y, a e he p epa a ion o osma inic acid, sage and sa o y nanopa icles.
Pa icula ly e iden we e he phenolic g oup bands (1360 and 1180 cm–1) in osma inic
acid nanopa icles and his is may be due o he highes amoun o osma inic acid in
hese nanopa icles, compa ing o he inside con en o osma inic acid in ex ac s and in o
he nanopa icles. This e ec was also epo ed by some au ho s ha de eloped a new
86
FTIR me hod o he cha ac e iza ion o osma inic acid in La andula o icinalis cul u es
(206). Ne e heless i mus be unde line ha his cha ac e is ic bands dec ease obse ed
in he nanopa icles by FTIR analysis, and he dec ease in an ioxidan ac i i y, may be
due o he pa ial e en ion o an ioxidan be o e hei comple e elease (conside ing he
heo e ical loadings unde s udied). This was documen ed o o he an ioxidan
nanopa icles s udies, such as essen ial oils (197). Also no new peaks appea ed in
nanoencapsula ion spec um, and chi osan, osma inic acid, sage and sa o y we e mixed
oge he physically wi hou any chemical eac ion (152).
87
Figu e 4. 6. Spec um o : I. (a). chi osan emp y nanopa icles; (b). osma inic acid in a
ee o m; (c). physical mix u e be ween chi osan unloaded nanopa icles and osma inic
acid (mixing a io 1:1); (d). osma inic acid encapsula ion in o chi osan nanopa icles.
II.(a). chi osan emp y nanopa icles; (b). sage in a ee o m; (c). physical mix u e be ween
94
whe e AbsABTS•+ deno es he ini ial abso bance o dilu ed ABTS•+, and Abs sample
deno es he abso bance o he sample by 6 min o eac ion. T iplica es o each sample
we e a e aged o gene a e each da um poin (which implies a o al o six eplica es pe
sample). The inal esul was exp essed as equi alen concen a ion o asco bic acid (in
g/L), using a calib a ion cu e.
5.1.5.2. Oxygen adical abso bance capaci y
The oxygen adical abso bance capaci y (ORAC) assay was employed o e alua e he
an ioxidan po en ial o chi osan-an ioxidan nanopa icles as desc ibed in p e iously
epo s (209). All eac ion mix u es we e p epa ed in duplica e, and a leas h ee
independen measu es we e pe o med o each expe imen . ORAC- luo escein (FL)
alues we e exp essed in µmol olox equi alen pe mg hyd olyzed o an ioxidan , as
pu posed elsewhe e (210).
5.1.6. S a is ical analysis
S a is ical analysis was pe o med using IBM SPSS S a is ics 19.0.0 (Illinois, USA). The
one-way analysis o a iance (ANOVA) was used wi h Sche é pos hoc es compa ison
o g oups wi h no mal dis ibu ion, and Mann-Whi ney es o g oups wi h non-no mal
dis ibu ion. Di e ences we e conside ed o be signi ican a a le el o P < 0.05.
5.2. Resul s and discussion
5.2.1. An ioxidan ac i i y measu emen
Rosma inic acid is phenolic compound, wi h many bene icial unc ionali ies and gene ally
admi ed as a ee adical sca enge (125). I s high biological ac i i y is pa icula ly ela ed
o i s wo ca echol moie ies. Ca echol is an impo an sub-s uc u e o he po en
an ioxidan ac i i y o phenolic an ioxidan s (125). The gene al an ioxidan mechanism o

95
phenolic compounds is hough o be di ided in o wo s ages: adical ca ching s age and
adical conclusion s age (126). Fo bo h me hods he an ioxidan ac i i y was es ed
acco ding o he an ioxidan ac i i y o osma inic acid, ei he in ee solu ion o
encapsula ed, bo h in ex ac s o in a ee o m. Fo bo h me hods no signi ican
di e ences we e ound o an ioxidan ac i i y be ween he all nano o mula ions, be o e
and a e lyophiliza ion p ocesses (P > 0.05), excep o sa o y encapsula ion. Fo hese
nanopa icles a dec ease in he an ioxidan ac i i y was obse ed a e lyophiliza ion,
especially e iden in ORAC me hod. Fo he loading concen a ions o 5 o 15%, no
an ioxidan ac i i y was ound by he ABTS o ORAC me hods. This means ha he
an ioxidan ac i i y was clea ly comp omised o hese low heo e ical loadings.
Conside ing he bo h ex ac s and osma inic acid nanopa icles, he highes an ioxidan
ac i i y was co ela ed o he highes an ioxidan s concen a ions (o highe loading alue)
(Table 5.1). Compa ing osma inic acid, sage and sa o y in a ee o m, i can be easily
obse ed ha osma inic acid has he highes an ioxidan ac i i y, ollowed by sage ha
has he highes con en o osma inic acid (10%) and hen sa o y wi h only (5%) o
osma inic acid con en . By ABTS me hod, compa ing he an ioxidan ac i i y be o e and
a e he encapsula ion p ocess, i was clea a dec ease in he an ioxidan ac i i y a e he
encapsula ion. This p oo s ha he an ioxidan ac i i y we e dec ease due o he pa ial
en apmen e ec o he compounds. None heless he pa icles s ill demons a ed good
an ioxidan ac i i y. This was in acco dance o o he p e iously epo s ha ha e showed
he same good an ioxidan e ec o T olox in chi osan nanopa icles (211). O he good
esul s we e ob ained o he chi osan encapsula ion o idebenone an ioxidan (201).
Fu he mo e and by ORAC, which is luo ime ic assay and a mo e sensi i e one, i can be
easily obse ed ha a e he encapsula ion p ocess he an ioxidan ac i i y was e en
lowe , han he esul s achie ed by ABTS (Table 5.1). These esul s we e consis en wi h
p e iously s udies wi h he encapsula ion o que ce in and u in (212). This s ill may be
due o he nanopa icles en apmen o osma inic acid, in o he way because he
an ioxidan was no comple ely eleased. This pa ial e en ion means ha he compounds
will ake mo e ime o build up hei speci ic ac i i y. Ne e heless he nanosys ems wi h
his en apmen e ec s ill ha e good an ioxidan ac i i y. Al hough e en i he nanosys em
an ioxidan ac i i y was lowe han he unloaded compounds, i is well known ha
nanoca ie s p o ec s he an ioxidan s o deg ada ion by biological and enzyma ic luids,
inc easing hei bioa ailabili y. The d ug elease can also be op imized o be e en longe ,
p olonged and con olled in ime conside ing he pu pose applica ion o his nanopa icles
(187). This makes he nanoencapsula ion ad an ageous and necessa y.
96
Table 5. 1. An ioxidan ac i i y measu emen s by ABTS and ORAC, conside ing he 50%
loading (m/m) sage and sa o y nanopa icles; and 40% loading (m/m) o and osma inic
acid nanopa icles o (n = 3).
ABTS x
ORAC y
(eq [Asc. Ac.]g/L)/g ex ac
(µmol/eq T olox)/g ex ac
F esh
Nanopa icles
Lyophilized
Nanopa icles
F esh
Nanopa icles
Lyophilized
Nanopa icles
Nano I
Rosma inic
acid
0.0348 ± 0.0050 a
0.0554 ± 0.0139 a
4.8374 ± 0.1719 b
3.6520 ± 0.1770 b
Sage
0.0537 ± 0.0015 c
0.0440 ± 0.0029 c
0.6227 ± 0.0901 d
0.4251 ± 0.0069 d
Sa o y
0.0828 ± 0.0102 e
0.0378 ± 0.0015
1.5315 ± 0.2784 g
0.4526 ± 0.0087 h
F ee II
Rosma inic
acid
0.0917 ± 0.0018
34.1218 ± 2.5733
Sage
0.1621 ± 0.0470
19.5924 ± 1.9791
Sa o y
0.1410 ± 0.0087
16.8117 ± 1.3605
No e: The esul s we e gi en as mean o iplica e samples, each wi h h ee measu emen s. The
same le e s, in he same line indica e ha no signi ican di e ences we e obse ed be ween he
esh and eeze-d ied p ocess (P > 0.05). The alues a e signi ican ly di e en (P > 0.05) o he
an ioxidan me hods (x, y) and o he encapsula ion p ocess (I, II).
Conside ing he same loading concen a ions o he osma inic acid and he ex ac s
nanopa icles i was clea ha osma inic acid in he ex ac s nanopa icles was in lowe
concen a ion han when was pu ely encapsula ed. Rosma inic acid nanopa icles (40%
loading), wi h 50% e iciency means encapsula e, 0.4 mg/mL o osma inic acid.
Ne e heless, sage o sa o y nanopa icles (50% loading), wi h almos 100% associa ion
e iciency (Table 4.2 – chap e 4), he nanosys em encapsula e 1 mg/mL o ex ac , bu
only 10 and 5% o osma inic acid (Table 4.2 – chap e 4), o sage and sa o y,
espec i ely. I is also known and documen ed by ou g oup (179), ha hese c ude
ex ac s (sage and sa o y) a e complex na u al ma ixes wi h di e en an ioxidan con en
(as P o oca echuic acid, couma ic acid, gallic acid, ca eic acid, e ulic acid, na ingenin,
que ce in, iso hamne in, chlo ogenic acid, p unin, isoo ien in, que ci in and u in).
Ne e heless when na u al ex ac s we e encapsula ed, he pH alue was o allow he
g ea amoun o osma inic acid in e ac ion wi h chi osan solu ion, and hen he success o
he encapsula ion. This means ha some phenolic compounds we e p esen in he
ex ac s, bu we e no nega i ely cha ged a o ma ion medium pH, may be los in
encapsula ion p ocedu e. Howe e , o he an ioxidan compounds in ex ac nanopa icles
ha ha e a close pKa o osma inic acid we e encapsula ed adding a syne gic an ioxidan
97
ac i i y. This jus i ies ha wi h lowe osma inic acid concen a ions, he ex ac s ha e he
same an ioxidan ac i i y, being good ehicles o osma inic acid and wi h good
an ioxidan syne gic pe o mance. Fu he mo e i was impo an o unde line ha
osma inic acid-nanopa icles can encapsula e wi h only 50% o associa ion e iciency,
which means a huge was e o he compound. This could ep esen ha a his momen ,
o all he es s made o chi osan nanopa icles wi h sage o sa o y, hey seem o be
good ehicles o osma inic acid inco po a ion and ep esen a mo e economically
p ocess, han nanopa icles wi h osma inic acid pu e. Ano he ad an age could be added
wi h he inclusion o biological ac i i ies o o he na u al compounds ha we e
inco po a ed a his pH alue in he nanopa icles. Howe e , in i o es s mus be done in
o de o gua an ee ha all he biological ac i i ies o he ex ac and o osma inic acid
we e main ained.
5.3. Conclusion
In his s udy, chi osan nanopa icles inco po a ing osma inic acid, sage and sa o y we e
p epa ed and cha ac e ized in o de o ensu e he highes an ioxidan ac i i y
pe o mance.
The bes an ioxidan ac i i y esul s we e ob ained o osma inic nanopa icles by ORAC
me hod a e lyophiliza ion, ca. 3.6520 ± 0.1770 µmol/eq T olox. None hless, sage and
sa o y showed o be good ehicles o osma inic acid ega ding he an ioxidan ac i i y
ou line. A e nanopa icles lyophiliza ion he ob ained alues we e: 0.4251 ± 0.0069 and
0.4526 ± 0.0087 µmol/eq T olox, o bo h sage and sa o y nanopa icles, espec i ely.
Ne e heless, i is impo an o unde line ha he c ude ex ac s ha e a lowe
concen a ion o osma inic acid, and he good an ioxidan ac i i y esul s sugges ed ha
o he syne gic compounds may be also encapsula ed. Besides a lowe an ioxidan ac i i y
was obse ed in he nanopa icles compa ing o he ee compounds due o he pa ial
en apmen e ec o he compounds, chi osan-based nanopa icles s ill main ained he
osma inic acid good an ioxidan ac i i y pe o mance and also allowing i s p o ec ion and
bes con ol i s p o ile elease. These esul s unde line he pha maceu ical po en ial o
chi osan-based nanosys ems o an ioxidan s deli e y.
98
99
PART V
“Science isn’ abou why…i ’s abou why no ?”
Unknown

100
101
Abs ac
In oduc ion: In i o assays a e c ucial o mimic biological condi ions and p edic
nanopa icles cy o oxici y, mucoadhesion and he encapsula ed bioac i e pe meabili y
p o iles. Neu oin lamma ion is a biological condi ion in ima e ela ed o he glaucoma
pa hophysiology, inc easing he eac i i y o mic oglia and he elease o p o-in lamma o y
media o s.
Objec i e: The goal o he s udy was o es he sa e y pe o mance, mucoadhesi eness
and pe meabili y o osma inic acid encapsula ed in o he a o emen ioned nanopa icles
owa ds ocula cell-based models, and p edic hei po en ial o p e en oxida i e eye
diseases. None heless, i was also in end o e alua e he e ec o osma inic acid in
e inal p o-in lamma o y con ol and in e i i may con e neu op o ec ion o he e ina in
an animal model o I-R.
Me hodology: Chi osan nanopa icles we e e alua ed conside ing mucoadhesi eness and
hei sa e y pe o mance and cell pe meabili y by in i o es s using ARPE-19 and HCE-T
monolaye cell lines. In i o assays we e pe o med injec ing in a i eally osma inic acid
in an I-R model, he osma inic acid neu op o ec ion was u he e alua ed by
elec o e inog ams (ERG) and immunohis ochemis y.
Resul s: Nanopa icles p e iously cha ac e ized demons a ed o be sa e wi hou ele an
cy o oxici y agains ARPE-19 and HCE-T cell lines, wi h no i i ancy o he eye. The
pe meabili y s udy in HCE monolaye cell line showed an appa en pe meabili y coe icien
Papp o 3.41 ± 0.99 x 10-5 and 3.24 ± 0.79 x 10-5 cm/s o osma inic acid loaded chi osan
nanopa icles and ee in solu ion, espec i ely. In ARPE-19 monolaye cell line he Papp
we e 3.39 ± 0.18 x 10-5 and 3.60 ± 0.05 x 10-5 cm/s o osma inic acid loaded chi osan
nanopa icles and ee in solu ion, espec i ely. Conside ing he mucin pa icle me hod,
nanopa icles indica e mucoadhesi e p op ie ies. ERG and immunohis ochemis y
showed ha a his concen a ion, by in a i eal injec ion, osma inic acid did no ha e a
e ina p o ec i e e ec in I-R s udied model.
102
Conclusion: The na u al nanopa icles de eloped in his s udy demons a ed o be in i o
p omising d ug deli e y sys ems o ocula applica ion. None heless, he in i o esul s
we e no e ec i e ega ding e ina neu op o ec ion, which may be due o he acu e I-R
model, which leads o se e e p o-in lamma o y damages ha an ioxidan s a e no able o
e e wi h a single injec ion. Despi e, his an ioxidan s pha maco he apy may be c ucial
o ocula diseases p ophylaxis.
103
CHAPTER 6 - In i o e alua ion o cy o oxici y,
mucoadhesion and ocula pe meabili y o
osma inic acid in o chi osan-based nanopa icles
110
6.1.6.1. Cy o oxici y es using cho ioallan oic memb ane
To e alua e he cy o oxici y and biocompa ibili y o ex ac s and osma inic acid-con aining
nanopa icles, Hen's Egg Tes s (HETs) we e pe o med on he cho ioallan oic memb ane
(CAM) as p e iously desc ibed (227). HET-CAM es me hod was used o he de ec ion o
ocula co osi es and se e e i i an s, as de ined by he U.S. En i onmen al P o ec ion
Agency (EPA 1996), he Eu opean Union (EU; EU 2001), and in he Uni ed Na ions
Globally Ha monized Sys em (GHS) o Classi ica ion and Labelling o Chemicals (UN
2003). Fe ile hen’s eggs a 10 days o incuba ion a 37 °C, ob ained om Gua a es
Gua abi a A es L da, we e used in he es s. Fi e eggs we e used o each nanopa icles
solu ion assay. A e 10 days o incuba ion, he egg shell abo e he ai space was
emo ed. The exposed memb ane was mois ened wi h a d op o 0.9% physiological
saline and he saline was emo ed, unco e ing he chick emb yo cho ioallan oic
memb ane (CAM). An aliquo o 200 μL o nanopa icles solu ion was applied on he CAM.
All assays we e epea ed i e imes. Signs o asocons ic ion, hemo hage and
coagula ion o 5 min we e obse ed e alua e he po en ial o i i a ion acco ding o he
me hod o HET-CAM. The ime (in seconds) a which he indica ed p ocesses began we e
applied in Equa ion (228). The ime (in seconds) a which he indica ed p ocesses began
we e applied in Equa ion (228):
𝐻𝑒𝑡− 𝐶𝑎𝑚 (𝐼𝑆)=(301−ℎ)×5
300 + (301−𝑣)×7
300 + (301−𝑐)×9
300
A e applica ion o he o mula abo e, i was possible o quan i y he obse ed po en ial
o i i a ion (i i a ion sco e-IS) and o ob ain means and s anda d de ia ions o he
analysis as ollows: 0-0.9 no i i a ion, 1-4.9 sligh i i a ion, 5-8.9 mode a e i i a ion and 9-
21 se e e i i a ion (228). All p ocedu es wi h chicken eggs we e ollowed by he
egula ions and p ocedu es o handling o human o animal ma e ials.

111
6.1.7. Pe meabili y s udies
6.1.7.1. Cell monolaye s cul u e
Immo aliza ion o human co neal epi helium (HCE) and e inal pigmen epi helium
(ARPE) cells ha e been desc ibed ea lie . Polyca bona e T answell® cell cul u e il e s
(Co ning, 3 μm, 6 wells, USA) we e used o pe meabili y assays. Suspension o HCE and
ARPE cells we e seeded on o he il e s a a concen a ion o 200.000 cells/cm2. The cells
we e g own a 37 °C in humidi ied ai wi h 5% o CO2, in s anda d cul u e medium in
apical chambe o 21 o 30 days un il he cells we e con luen . The cul u e DMEM
medium was eplaced e e y wo days.
6.1.7.2. T ansepi helial elec ical esis ance
T ansepi helial elec ical esis ance (TEER) was measu ed a di e en phases o cell
g ow h (E om; Wo ld P ecision Ins umen s, Sa aso a, FL), as an indica o o epi helial
di e en ia ion and epi helial igh ness. TEER da a we e co ec ed o low-backg ound
TEER by using a blank il e con aining he possible coa ing ma e ials and cul u e
medium. A he end o each pe meabili y expe imen , TEER was measu ed o de ec he
condi ion o he cells.
6.1.7.3. Pe mea ion s udies in cell monolaye s
The pe mea ion s udy wi h di e en solu ions was ini ia ed by washing wi h HBSS (1x)
liquid (wi hou calcium and magnesium) bo h he basola e al and apical side one ime and
hen adding 2.5 mL o HBSS o he basola e al side ( ecei e side) and 1.5 mL o HBSS o
he apical side (dono side). A di e en ime poin s, du ing 60 minu es, aliquo s o 100 µL
we e wi hd awn om he ecei e chambe and eplaced wi h an equal olume o blank
medium. TEER was measu e a each ime and he pla es we e incuba ed wi h he sample
112
a 37 ºC o sum o he es imes. Papp was calcula ed om he measu emen o he low
a e o insulin om he dono o he accep o chambe s:
P𝑎𝑝𝑝 (𝑐𝑚 𝑠)= 𝑑𝑄 𝑑𝑡
⁄ (𝐴 × 𝐶0)
⁄
whe e, dQ is he o al amoun o pe mea ed osma inic acid (mg), A is he di usion a ea
(cm2), C0 is he ini ial concen a ion o osma inic acid (mg/mL), and d is he ime o
expe imen in seconds (s). The coe icien dQ/d ep esen s he s eady-s a e lux o
osma inic acid ac oss he monolaye .
6.1.7.4. High pe o mance liquid ch oma og aphy analysis
The HPLC me hod was de eloped and alida ed, as p e iously desc ibed in chap e 3.
B ie ly, aliquo s o 100 µL we e injec ed a HPLC o quan i y which quan i y concen a ion
pe mea es o he basola e al side o he pla es. All HPLC uns we e pe o med using a
Wa e s Se ies 600 HPLC and esul s we e acqui ed and p ocessed wi h Empowe ®
So wa e 2002 o da a acquisi ion (Mild o d MA, USA). HPLC analysis was conduc ed by
using a No a-Pack® RP C18 column (250 x 4.6 mm i.d., 5 µm pa icle size and 125 Å
po e size) om Wa e s. Ch oma og aphic analysis was pe o med in g adien mode. The
mobile phase consis ed o me hanol: o mic acid: wa e UP in he a io 92.5:2.5:5 ( / ).
S a iona y phase was made wi h he same componen s in he a io o 5:2.5:92.5,
espec i ely. The phases we e il a ed h ough 0.22 µm il e and degassed. Eluen was
pumped a a low a e o 0.75 mL/min, he injec ion olume was 20 μL and de ec ion
wa eleng h was 280 nm.
6.1.8. S a is ical analysis
S a is ical analysis was pe o med using IBM SPSS S a is ics 19.0.0 (Illinois, USA). The
one-way analysis o a iance (ANOVA) was used wi h Sche é pos hoc es compa ison
o g oups wi h no mal dis ibu ion, and Mann-Whi ney es o g oups wi h non-no mal
dis ibu ion. Di e ences we e conside ed o be signi ican a a le el o P < 0.05.
113
6.2. Resul s and discussion
6.2.1. Pa icle size, polydispe si y and ze a po en ial
The pa icle size and mean size dis ibu ion a e undamen al ea u es ha in luence he in
i o dis ibu ion, biological a e, oxici y and he a ge ing abili y o nanopa icles
con aining he apeu ic d ugs (191). I is also known ha he highes alue o ze a po en ial
ep esen s he g ea e elec os a ic epulsi e in e ac ions among he pa icles. Ze a
po en ial alues o ± 30 mV indica es ha he colloidal sys ems a e s able in ime and ha
amine g oups o chi osan a e on he su ace (193). The de eloped nanopa icles anged
om 200-300 nm in size and ze a po en ial we e a ound 20-30 mV (Table 6.1). I is
desc ibed ha o ul a ine pa icles he induc ion o ROS, OS, in lamma ion and
ascula u e a e a isk (229). In his sense i can be conside ed ha he pa icles o his
s udy will ha e no such ha m ul e ec s. Mo eo e , he esul s a e in ag eemen wi h
simila nanopa icles con aining osma inic acid we ecen ly de eloped (230) and wi h
o he ex ac s, also encapsula ed in chi osan nanopa icles (193). The esul s did no
show signi ican di e ences (P > 0.05) in size be ween he osma inic acid and he wo
c ude ex ac s, as p e iously desc ibed (190, 196). This may be due o he high osma inic
acid con en in ex ac s composi ion 10 and 5% in sage and sa o y, espec i ely (196).
Thus he ex ac s unde s udy a e p omising ehicles o osma inic acid nano-
inco po a ion, as p e iously documen ed (190, 196). The size ob ained is also in
ag eemen wi h ocula d ug deli e y demands, as pa icles wi h size ≤ 200 nm we e
obse ed o each he e ina, i eous and abecula meshwo k (231) and ha e mo e
i eal hal -li e compa ed o huge nanopa icles (232). The ob ained nanopa icles also
showed alues o polydispe si y be ween 0.1 and 0.2 co esponding o a na ow
dis ibu ion and monodispe sed pa icles. These esul s a e in acco dance o o he wo ks
using simila an ioxidan nanopa icles (194).
114
Table 6. 1. A e age hyd odynamic diame e (Z), polydispe si y index (PdI) and ze a
po en ial o chi osan nanopa icles loaded osma inic acid, sage and sa o y.
Nano
Z-a e age
(nm)
PdI
Ze a po en ial
(mV)
Associa ion
e iciency
(%)
Loading
capaci y
(%)
RA
280.0 ± 16.0 a
0.201 ± 0.091 b
30.1 ± 1.8 c
60.2 ± 1.3 d
5.3 ± 0.4
Sage
302.4 ± 18.2 a
0.288 ± 0.074 b
27.5 ± 0.9 c
96.8 ± 0.2 e
8.1 ± 0.6 g
Sa o y
298.3 ± 20.8 a
0.214 ± 0.085 b
28.2 ± 2.2 c
98.0 ± 0.3 e
7.8 ± 0.2 g
No e: Values we e means o iplica e samples ± s anda d de ia ion; a,b,c,d,e, ,g means wi hin he
same column, labelled wi h he same le e , we e no s a is ically di e en om each o he (P >
0.05).
6.2.2. Associa ion e iciency and loading capaci y
Chi osan can in e ac wi h he nega i ely cha ged TPP, o ming in e - and in a-molecula
c oss-linkages, yielding ionically c oss-linked chi osan nanopa icles in acidic media (pKa
6.5) (36). I is also known ha he in e - and in a-molecula linkages c ea ed be ween
TPP and he posi i ely cha ged amine g oups o chi osan we e esponsible o he
success o he gela ion p ocess (188). Among he di e en sou ces o osma inic acid
encapsula ed in o chi osan nanopa icles, i was obse ed di e en loading capaci y
be ween ee d ug and ex ac s (Table 6.1). This may be co ela ed o he lowe ini ial
amoun in osma inic acid used o p epa e nanocomplexes, as p e iously desc ibed o
insulin encapsula ion in o chi osan-algina e iono opic nanopa icles (233). The esul s
we e in line wi h p e ious da a o ionic gela ion chi osan nanopa icles (234, 235). Highe
associa ion e iciency was ound o osma inic acid en apmen in ex ac s o sage and
sa o y (wi h no signi ican di e ences obse ed be ween hem (P > 0.05), han in pu e
osma inic acid en apmen in chi osan nanopa icles. The esul s we e on he ow wi h
o he chi osan idebenone and ilex pa aguensis encapsula ion (193, 201). O he s udies
showed simila associa ion e iciency o o he phenolic compound, such as ca hechin
(198) and epigalloca echin galla e (199). The esul s also exhibi ed highe associa ion
e iciency han he ob ained o que ce in encapsula ion in PLA nanopa icles (i.e. 40%)
(152). Ne e heless, highe associa ion e iciency o osma inic acid nanopa icles we e
p e ious desc ibed in solid-lipid-nanopa icles (230). Ne e heless, highe associa ion
e iciency o osma inic acid nanopa icles we e p e ious desc ibed in solid-lipid-
115
nanopa icles (230). The e o e, he associa ion e iciency was highly dependen on he
nanosys em used, as well as om he loading and osma inic acid amoun u ilized in o he
nanopa icles.
6.2.3. Mucoadhesion p op ie ies e alua ion by mucin in e ac ion me hod
Ocula mucosa may a ec he s abili y chi osan nanopa icles in he p esence o mucus
componen s (2). Mucus consis s in a he e ogeneous idimensional ne wo k, being
basically composed o a mucin ibe s ne wo k, c ea ing an endless sys em o canals in
which pa icles can di use and/o be e ained (236, 237). Chi osan is a mucoadhesi e
polyme ha may inc ease esidence ime and in ima e con ac o he deli e y ehicle wi h
he mucosa, consequen ly inc easing he d ug bioa ailabili y, such p op ie ies we e well
documen ed in li e a u e (168). The chi osan adhesion mechanism is mainly associa ed o
he elec os a ic in e ac ions es ablished be ween p o ona ed amine g oups o
mucoadhesi e chi osan and nega i ely cha ged g oups o mucin (238). In his s udy, he
nanopa icle-mucin in e ac ion was de e mined measu ing he amoun o mucin ha
a aches he nanopa icles (239). The deg ee o adso p ion o nanopa icles/mucin
pa icles can be de e mined by he a ia ions in size (240), ze a po en ial (241) o
elec opho e ic mobili y (242) o o med complexes wi h mucosal luids, in pa icula wi h
mucin. Size can in luence he di usion o d ug ca ie s h ough he mucin mesh ha
composes mucus luids, being he op imal ange be ween 200 and 500 nm o enhance
di usion (243). Highe o smalle diame e s may dec ease anspo h ough he mucus
laye . This is in con as o he p e ailing belie , demons a e ha la ge nanopa icles, can
apidly pene a e physiological human mucus, and ha la ge nanopa icles can be used
o mucosal d ug deli e y (243). Fo his pu pose, osma inic acid con aining nanopa icles
we e e alua ed, since chi osan was he only esponsible o he mucoadhesion esul s
and osma inic acid was he p inciple ac i e o all he o mula ions used. As depic ed in
Table 6.2, he osma inic acid nanopa icles ob ained in his s udy we e in op imal ange
men ioned, o he h ee pHs used. The in luence o pH alues was in ended o
demons a e ha he pa icles we e mucoadhesi e in di e en condi ions, a he no mal
ionic gela ion pH (5.8), a an in lamma ion pH (5.0) and in homeos asis pH (7.4). The
inc eased size a e mucin incuba ion sugges s ha nanopa icles-mucin in e ac ion we e
o ming mic oagg ega es, and dec ease in ze a po en ial alues was p obably due o he
elec os a ic in e ac ion be ween posi i e cha ged o chi osan and anionic mucin (241).

116
S ong abili y o hose nanopa icles o in e ac wi h mucin h ough elec os a ic o ces
we e obse ed, highligh ing hei po en ial as mucoadhesi e ca ie s (226). O he s udy
demons a ed he e ec o low, medium and high mola mass chi osan in coa ed poly-
cap olac one (PCL) nanopa icles (238). The esul s we e acco ding o mucoadhesi e
nanopa icles made o hiola ed qua e na y chi osan c osslinked wi h hyalu onan (244).
Table 6. 2. A e age hyd odynamic diame e (Z), polydispe si y index (PdI) and ze a
po en ial o chi osan nanopa icles loaded osma inic acid be o e and a e mucin
in e ac ion (n = 3).
Nanopa icles
pH
Z-a e age (nm)
PdI
Ze a po en ial (mV)
Rosma inic acid
5.8
236.0 ± 7.1a
0.719 ± 0.036
40.1 ± 0.8 c
Rosma inic acid + Mucin
5.0
488.2 ± 30.5 b
0.619 ± 0.049
22.5 ± 0.9 d
Rosma inic acid + Mucin
7.4
414.1 ± 32.3 b
0.511 ± 0.014
23.1 ± 0.6 d
No e: Values we e means o iplica e samples ± s anda d de ia ion; a, b, c, d means wi hin he same
column, labelled wi h he same le e , we e no s a is ically di e en om each o he (P > 0.05).
6.2.4. Cell iabili y s udies
In o de o e alua e any po en ial cy o oxici y o osma inic acid, sage and sa o y on
ARPE-19 and HCE-T cell lines, pu e osma inic acid and ex ac s-loaded chi osan
nanopa icles we e es ed o 4 and 24 h. The e ec o nanopa icles on memb ane
in eg i y was measu ed by he LDH enzyme elease assay and he e ec on cell iabili y
was measu ed using he MTT con e sion assay. In i o cy o oxici y esul s we e
p esen ed in (Figu e 6.1). Resul s showed ha a e 4 h he cy o oxici y was below 10%
o he es ed concen a ion ange, o bo h cells lines. Mo eo e , he e we e no signi ican
di e ences (P > 0.05) be ween he 4 and 24 h ( es ing ime) conside ing all o mula ions
and di e en cell lines.
117
No e: NR, R - osma inic acid loaded and unloaded chi osan nanopa icles, espec i ely; NSL, SL
– Sal ia o icinalis loaded and unloaded chi osan nanopa icles, espec i ely; NSG, SG – Sa u eja
mon ana loaded and unloaded chi osan nanopa icles, espec i ely.
Figu e 6. 1. E ec o osma inic acid, sage and sa o y-loaded chi osan nanopa icles on
cell cy o oxici y o ARPE (A, C and E) and HCE (B, D and F) cell lines a e 4 h (black ba )
and 24 h (whi e ba ) o incuba ion. DMEM+cells and DMSO we e used as con ols. The
o mula ion concen a ion used was displayed in he ables, ela i ely o osma inic acid
(A, B), sage (C, D) and sa o y (E, F) ( esul s we e he mean o 6 eplica es, ba s
ep esen s anda d de ia ion).
118
Resul s o cell iabili y o osma inic acid, sage and sa o y loaded chi osan nanopa icles
ob ained om he MTT es we e shown in Figu e 6.2. These we e in a good co ela ion
wi h hose om he LDH assay. An ioxidan /chi osan nanopa icles p esen ed a good
p o ile in e ms o cell iabili y o ARPE-19 and HCE-T cells. The esul s showed ha
hese nanopa icles we e no oxic o he cells a concen a ions below 1 mg/mL. The
esul s we e in line wi h chi osan cell sa e y pe o mance ega ding i s use as d ug
deli e y sys ems and da a show ha i was comple ely sa e wi h no- oxici y e ec upon
cell lines since chi osan can in e ac wi h cell memb anes and be up ake wi h no
cy o oxici y and cell colla e al damages. The esul s we e in line wi h p e ious s udies on
he use o chi osan in d ug deli e y sys ems, (40) ega ding i s non- oxici y, good cell
memb ane in e ac ion and cellula up ake wi h no colla e al damages. Mo eo e , o he
s udies demons a ed ha chi osan nanopa icles besides being non- oxic, may ha e
p o ec i e e ec s on cell lines (245-247).
119
No e: NR, R - osma inic acid loaded and unloaded chi osan nanopa icles, espec i ely; NSL, SL
– Sal ia o icinalis loaded and unloaded chi osan nanopa icles, espec i ely; NSG, SG – Sa u eja
mon ana loaded and unloaded chi osan nanopa icles, espec i ely.
Figu e 6. 2. E ec o osma inic acid, sage and sa o y-loaded chi osan nanopa icles on
iabili y o ARPE (A, C and E) and HCE (B, D and F) cell lines a e 4 h (black ba ) and 24
h (whi e ba ) o incuba ion. DMEM+cells and DMSO we e used as con ols. The
o mula ion concen a ion used was displayed in he ables, ela i ely o osma inic acid