Advances on the formulation of proteins using nanotechnologies

Accep ed Manusc ip
Ad ances on he o mula ion o p o eins using nano echnologies
I ene San alices, And ea Gonella, Dolo es To es, Ma ía José Alonso
PII: S1773-2247(17)30251-4
DOI: 10.1016/j.jdds .2017.06.018
Re e ence: JDDST 416
To appea in: Jou nal o D ug Deli e y Science and Technology
Recei ed Da e: 23 Ma ch 2017
Re ised Da e: 22 June 2017
Accep ed Da e: 23 June 2017
Please ci e his a icle as: I. San alices, A. Gonella, D. To es, Ma í.José. Alonso, Ad ances on he
o mula ion o p o eins using nano echnologies, Jou nal o D ug Deli e y Science and Technology
(2017), doi: 10.1016/j.jdds .2017.06.018.
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Ad ances on he o mula ion o p o eins
1
using nano echnologies
2
I ene San alices
a*
, And ea Gonella
a*
, Dolo es To es
b
, Ma ía José Alonso
a,b#
3
4
a
Cen e o Resea ch in Molecula Medicine and Ch onic Diseases (CIMUS), Campus Vida 5
Uni e si y o San iago de Compos ela, San iago de Compos ela 15782, Spain. 6
7
b
Depa men o Pha maceu ics and Pha maceu ical Technology, School o Pha macy, Campus 8
Vida, Uni e si y o San iago de Compos ela, San iago de Compos ela 15782, Spain. 9
10
*These au ho s con ibu ed equally o his wo k. 11
12
#
Co esponding au ho e-mail add ess: ma [email protected] 13
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Table o con en s
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1. In oduc ion 36
2. Fo mula ion echnologies (nanoca ie s, p epa a ion echniques, cha ac e iza ion, d ug 37
loading and elease p o ile) 38
2.a. Lipid-based nanoca ie s 39
2.a.1. Liposomes 40
2.a.2. Solid Lipid Nanopa icles (SLN) 41
2.a.3. Mic oemulsions and nanoemulsions 42
2.a.4. Nanocapsules (NCs) 43
2.b. Polyme -based nanoca ie s 44
2.b.1. Polyes e s-based nanoca ie s 45
2.b.2. Ac ylic polyme s-based pa icles 46
2.b.3. Polysaccha ide-based pa icles: 47
2.b.4. P o ein-based pa icles: 48
3. Cu en s a us o pep ide/p o ein-loaded nano echnologies 49
4. Conclusions 50
51
52
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55
56
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Abs ac
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The apeu ic p o eins and pep ides a e e y a ac i e om he pha maceu ical poin o iew due 77
o hei high po ency and selec i i y. None heless, hei ins abili y and low bioa ailabili y make 78
hei adminis a ion h ough non pa en e al ou es e y di icul , a ac ha hampe s hei 79
e icien exploi a ion in he apeu ics. Since he 70´s, signi ican amoun o esea ch in he a ea o 80
d ug deli e y and nano echnology has been done wi h he inal goal o o e coming hose hu dles. 81
In pa icula , biodeg adable and biocompa ible lipid and polyme -based nanoca ie s ha e 82
eme ged as p omising deli e y pla o ms o enable he adminis a ion o p o eins and pep ides. 83
This e iew p o ides an o e iew o he mos ly explo ed nano echnologies o da e in ended o 84
p oduce lipidic and polyme ic nanoca ie s o p o ein/pep ide deli e y. The basic p inciples o he 85
di e en echniques a e discussed, and he main ac o s in ol ed in he d ug associa ion and 86
elease, a e analyzed. Finally, a b ie o e iew o he po en ial applica ions o hese 87
p o ein/pep ide-loaded nanoca ie s, highligh ing he nanomedicines ha ha e eached he 88
ma ke o he clinical de elopmen phase, is p o ided. 89
90
Keywo ds: p o ein deli e y, pep ide deli e y, lipid o mula ion, polyme ic o mula ion, 91
nanocapsule, nanopa icle, liposome, mic oemulsion 92
93
G aphical abs ac :
94
95
*X- ay s uc u e o Human Recombinan insulin
.
Image om he RCSB PDB (www. csb.o g) o PDB ID 5E7W.
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1. In oduc ion
98
99
Du ing he las decades, impo an e o s ha e been o ien ed o he comme cializa ion o 100
he apeu ic p o eins and pep ides. Un o una ely, despi e he well-known ad an ages o hese 101
d ugs in e ms o po ency and selec i i y, hei exploi a ion is being limi ed by hei ins abili y, 102
es ic ed bioa ailabili y and in insic immunogenici y (specially o high molecula weigh 103
p o eins) [1]. These d aw-backs ha e s imula ed he esea ch in he a ea o d ug deli e y and 104
nano echnology wi h he inal goal o making he adminis a ion o hese powe ul d ugs mo e 105
e icien [2–4]. 106
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The possibili y o including pep ides and p o eins in nano ehicles ha a e able o p o ec and 108
deli e hem a he adequa e si e has gene a ed inc easing expec a ion du ing las decades (Fig. 1) 109
[5]. Liposomes we e he i s nanoca ie s p oposed o p o ein deli e y in he ea ly 70’s [6,7]. 110
Meanwhile, Speise and co-wo ke s in es iga ed he possibili y o encapsula e d ugs o an igens 111
in o polyac ylic nanopa icles using micelle polyme iza ion echniques [8]. A decade la e , 112
poly(alkylcyanoac yla e) nanocapsules we e p oposed as ca ie s o he o al adminis a ion o 113
insulin [9]. Finally, o e he 90’s Gasco e al. p oduced o he i s ime pep ide-loaded solid lipid 114
nanopa icles [10–12] and ou g oup pionee ed he de elopmen o nanopa icles made o PLGA 115
[13], PLA-PEG [14] and chi osan [15] o he deli e y o p o eins and an igens. As illus a ed in 116
Figu e 1, he in e es a ound he use o all hese nanoca ie s o p o ein/pep ide deli e y has 117
p og essi ely inc eased in he pas decades, being liposomes and polysaccha ide-based 118
nanopa icles he ones ecei ing he g ea es a en ion. No ewo hy, he use o ino ganic 119
nanopa icles in he pep ide/p o ein deli e y ield has g own-up du ing he pas decade, as well. In 120
pa icula , nanopa icles made o gold, i on oxide [16,17], calcium phospha e and silica likewise 121
ca bon nano ubes [18,19] ha e ecei ed a ce ain a en ion. Howe e , o e all, he endency has 122
been owa ds he use o biodeg adable and biocompa ible bioma e ials ha can o m 123
nanos uc u es based on iendly and easily scalable echniques. This endency is expec ed o 124
change he ansla ional p ospec i e o hese deli e y ehicles. Indeed, s ill nowadays he 125
de elopmen o e icacious and cos –e ec i e nano-based p o ein p oduc s emains a challenge 126
and his jus i ies he limi ed numbe o p o ein/pep ide-loaded nanopa icula e p oduc s in he 127
ma ke [20–22]. The necessi y o hese nanomedicines o exhibi impo an quali y a ibu es 128
such as signi ican d ug loading, main enance o he loaded pep ide/p o ein ac i i y and con olled 129
d ug elease, a e jus some examples o he bo lenecks o be o e came [23]. 130
131
Figu e 1. T end o epo ed expe imen al wo ks conce ning nanoca ie s o pep ide/p o ein deli e y om he 70’s up o
132
da e. Da a aken om Scopus (1971–2017) using p o ein/pep ide deli e y and he ype o sys em as sea ching c i e ia.
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ME: mic oemulsion; NCs: nanocapsules; NE: nanoemulsion; NPs: nanopa icles; SEDDS: sel -emulsi ied d ug deli e y
134
sys em; SMEDDS: sel -mic oemulsi ied d ug deli e y sys em; SNEDDS: sel -nanoemulsi ied d ug deli e y sys em; SLN:
135
solid lipid nanopa icles.
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137
This e iew aims o analyze he main echnologies employed un il oday o p oduce lipid and 138
polyme -based nanopa icula e ca ie s o pep ide/p o ein deli e y. Addi ionally, a b ie o e iew 139
o s a e o he a o he p o ein loading, p o ein s uc u al s abili y and elease p ope ies om 140
hese nanoca ie s, as well as, hei inal applica ions, is discussed. The analysis o he echnologies 141
o p oduce ino ganic pa icles and hei cha ac e iza ion was conside ed o be beyond he scope 142
o his e iew. 143
144
2. Fo mula ion echnologies
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146
2.a. Lipid-based nanoca ie s
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148
In he las decades, lipid-based nanoca ie s (Fig. 2) ha e eme ged as po en ial nanoca ie s o 149
mac omolecula deli e y. This has been mainly due o he abso p ion enhancing p ope ies o he 150
lipids and he nanoca ie ’s abili y o imp o e he d ug s abili y. Fu he mo e, he biocompa ible 151
cha ac e o hese bioma e ials and he low cos o he p oduc ion echniques ha e inc eased he 152
in e es in hese nanoca ie s [24]. Despi e o his, he inclusion o hyd ophilic mac omolecules 153
in o hese sys ems has been so a limi ed by hei solubili y. In o de o imp o e hei 154
inco po a ion in o hese sys ems, many inno a i e s a egies, which a e summa ized below, ha e 155
been desc ibed as p omising app oaches o he o mula ion o pep ide lipid-based deli e y 156
nanosys ems (Fig. 3). 157
158
Figu e 2. Illus a ion o he main lipid-based nanosys ems explo ed o p o ein/pep ide deli e y. Adap ed wi h pe mission
159
om [25].
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161
- Re e se micelliza ion. This s a egy in ol es he use o amphiphilic molecules able o sel -162
o ganize as e e se micelles exposing hei hyd ophobic chains o he ex e io and hei 163
hyd ophilic head g oups o he inne pa o he s uc u e [26]. This inne ca i y acili a es he 164
inco po a ion o hyd ophilic mac omolecules p io o i s inclusion in he inal sys em [27]. 165
166
- Double emulsion me hod. This echnique consis s on he o ma ion o a W/O emulsion in which 167
he hyd ophilic d ug is con ined wi hin i s in e nal aqueous phase p io o i s inclusion in he inal 168
sys em [28–30]. 169
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- Hyd ophobic ion pa ing. This app oach has been used o enhance he hyd ophobici y o he 171
d ug, he eby imp o ing i s lipid solubili y. I is based on he ionic complexa ion o a 172
pep ide/p o ein wi h a molecule, o en an amphiphilic compound, wi h an opposi e su ace cha ge 173
[11,31,32] o e en wi h complex s uc u es such as liposomes [33]. 174
175
- Hyd ophobic – hyd ophilic in e ac ions. This app oach in ol es he dispe sion o an aqueous 176
solu ion o he hyd ophilic d ug in o an amphiphilic compound, ollowed by he addi ion o he 177
o med dispe sion in o he oily phase [34,35]. 178
179
180
Figu e 3. Illus a ion o he main s a egies employed o imp o e he inco po a ion o hyd ophilic mac omolecules in o
181
lipid-based deli e y nanosys ems. Adap ed wi h pe mission om [25].
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183
2.a.1. Liposomes
184
185
Since hei disco e y in 1964 [36,37], liposomes ha e been he mos ex ensi ely d ug deli e y 186
ehicles in es iga ed. To da e, 13 liposome-based p oduc s ha e been app o ed o human use by 187
he FDA [25]. B ie ly, liposomes a e de ined as esicles wi h an aqueous co e in he inne ca i y, 188
su ounded by one o mo e bilaye s o amphiphilic phospholipids. Thei sizes ange om 20 nm (i 189
unilamella ) up o mic ons (i mul ilamella ) [38]. Among he wide a ie y o lipids, hose 190
amphiphilic able o sel -assembly, such as phospholipids, phospha idylglyce ol de i a i es and 191
bo h sa u a ed and unsa u a ed a y acids, a e he mos commonly used o p oducing liposomes 192
[25]. Addi ionally, i is also possible he inclusion o polyme s and su ac an s in o hei s uc u e 193
[39–42]. Finally, he use o special lipids has led o he o ma ion o nanos uc u es named as 194
a cheosomes (i.e., die he o e ae he lipids) [43] and niosomes (i.e., polyoxye hylene alkyl 195
e he s) [44], which we e supposed o acili a e he en apmen o pep ides and p o eins [45]. 196
197
2.a.1.a. P epa a ion echniques 198
199
O e all, he echnologies o p epa e liposomes a e ela i ely simila . The main di e ence among 200
he a ie y o echniques desc ibed so a elies on he way o d ying he lipids om he o ganic 201
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sol en s and ehyd a ing hem in aqueous media [46]. The main liposomes p epa a ion me hods 202
used o p o ein/pep ide associa ion a e hose desc ibed below. 203
204
i) Film hyd a ion 205
This echnique was in oduced by Bangham and cowo ke s o p oduce liposomes by he i s ime 206
(Fig. 4) [36,37]. This echnique in ol es he dissolu ion o he phospholipids in an o ganic sol en , 207
ollowed by he sol en e apo a ion and he deposi ion o he phospholipids o ming a lipid ilm. 208
Then, an aqueous solu ion con aining he p o ein is added o e he lipidic ilm o hyd a e i , 209
usually wi h he help o sonica ion, hus leading o he o ma ion o liposomes [47,48]. 210
211
212
Figu e 4. Schema ic iew o he ilm hyd a ion echnique o p oduce liposomes
213
214
ii) Re e se-phase e apo a ion 215
This echnique simply in ol es he o ma ion o e e se micelles by mixing an o ganic solu ion o 216
he phospholipids wi h a small olume o an aqueous phase con aining he pep ide/p o ein, 217
usually using sonica ion. The e apo a ion o he sol en esul s in he o ma ion o la ge 218
unilamella o mul ilamella liposomes (Fig. 5) [49]. 219
220
221
222
Figu e 5. Schema ic iew o he e e se-phase e apo a ion echnique o p oduce liposomes
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224
2.a.1.b. Cha ac e iza ion, pep ide/p o ein loading, ac i i y and elease p o ile 225
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- Pa icle size dis ibu ion: gene ally, a homogeniza ion s ep is necessa y in o de o ob ain a 227
na ow pa icle size dis ibu ion. The homogeniza ion o he sys em can be achie ed using 228
ex usion [50,51], eeze- hawing [52,53], dehyd a ion- ehyd a ion [54,55], sonica ion o high 229
p essu e. Likewise, he a io be ween he di e en componen s will in luence he inal liposomes 230
pa icle size dis ibu ion. 231
232
- Pep ide/p o ein loading and ac i i y: liposomes ha e he abili y o encapsula e hyd ophilic (in 233
he inne aqueous co e), lipophilic (wi hin he lipid bilaye ) o amphiphilic d ugs (pa i ioned 234
be ween he lipid bilaye and he aqueous co e) [56]. In gene al, he d i ing o ce o he 235
encapsula ion elies on he in e ac ion be ween he p o ein/pep ide and he lipids and also on he 236
bilaye igidi y. Fo example, liposomes wi h insulin associa ion e iciency (AE) alues a ying om 237
10 up o 90 % could be ob ained by changing he phospha idylcholine:phospha idyle hanol a io 238
[57]. To da e, a a ie y o pep ides and p o eins ha e been e icien ly en apped in o liposomes 239
using he p epa a ion me hods disclosed in Table 1. Un o una ely, he loading capaci y o he 240
esul ing o mula ions has no been desc ibed o has been low (< 1 %) [58]. The e o e, he loading 241
capaci y could be conside ed as a limi a ion o hese deli e y ca ie s. 242
243
Addi ionally, he loaded p o ein mus emain ac i e once encapsula ed in o he liposomes. The 244
sou ces o pep ide/p o ein ins abili y di e depending on he p oduc ion me hod conside ed, 245
being he ilm hyd a ion he less s ess ul o he in eg i y o he p o ein, e en i sonica ion could 246
a ec i s s uc u e [59]. On he o he hand, he e e se phase e apo a ion echnique di ec ly 247
exposes he pep ide/p o ein o o ganic sol en s, wi h he subsequen possibili y o su e ing 248
dena u a ion [59,60]. Homogeniza ion, ex usion and eeze haw cycles can also cause p o ein 249
dena u a ion/agg ega ion in bo h me hods [59]. The in eg i y o he loaded pep ide/p o ein has 250
been s udied using di e en me hods, such as elec opho esis-based echniques (e.g. Wes e n 251
blo , SDS-PAGE, e c.), p o ein ac i i y (pa icula ly i he encapsula ed p o ein is an enzyme) o 252
di ec ly h ough in i o expe imen s [48,57,59,61]. 253
254
- Pep ide/p o ein elease: he physicochemical p ope ies o he phospholipids a e known o 255
de e mine he luidi y o he lipid bilaye and, as a consequence, in luence he pep ide/p o ein 256
elease p o ile. In his sense, a mo e sus ained elease is ob ained when inc easing he igidi y o 257
he bilaye by he inclusion o choles e ol o long hyd ophobic chains in he liposome [62]. 258
S a egies o con ol he elease o pep ides/p o eins om liposomes, such as he su ace 259
modi ica ion wi h polye hylene glycol (PEG) o o he polyme s, as well as hei inclusion in o he 260
nanos uc u es, ha e been de eloped [58,63–66]. Fo example, a lowe insulin elease was 261
showed a e 4 hou s in simula ed in es inal luids om laye -by-laye coa ed liposomes (20 %) 262
compa ed o hose uncoa ed (60 %) [48]. 263
264
In conclusion, bo h ilm hyd a ion and e e se phase e apo a ion me hods a e sui able o 265
encapsula ing pep ides/p o eins in liposomes, allowing bo h, good associa ion e iciencies and 266
sus ained elease p o iles. The bilaye igidi y and he elec os a ic in e ac ions be ween he 267
pep ide and he liposomes componen s a e he main ac o s condi ioning he loading capaci y o 268
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As indica ed, he p o ein/pep ide s abili y is gene ally in luenced by he p esence o o ganic 416
sol en s (e.g. double emulsion/sol en e apo a ion o nanop ecipi a ion me hods), high shea 417
mechanical agi a ion and p essu e (e.g. HPH me hod), high empe a u es (e.g. 418
mic oemulsi ica ion/solidi ica ion and HPH me hods) o sonica ion p ocesses (double 419
emulsion/sol en e apo a ion me hod) [11,29,30,88]. The p o ein in eg i y and ac i i y has been 420
usually analyzed using he same echniques desc ibed o liposomes in he p e ious 2.a.1.b. 421
sec ion (i.e., SDS-PAGE, capilla y elec opho esis, enzyma ic assays and in i o s udies) [11,28–422
30,76]. 423
424
- Pep ide/p o ein elease: da a indica ed in Table 3 highligh he high a iabili y in he elease 425
p o iles obse ed o di e en pep ides/p o eins en apped in a a ie y o SLN [28,78,79]. 426
Al hough in some wo ks, no bu s elease was epo ed, no mally he e is a a iable amoun o 427
pep ide accumula ed a he O/W in e ace du ing he p oduc ion p ocess ha is eleased 428
p ema u ely [72]. This bu s e ec and he subsequen elease p o ile has been modula ed 429
ollowing speci ic o mula ion app oaches. In pa icula , he o e all elease p o ile is highly 430
dependen on he SLN composi ion, since i is mainly go e ned by he pep ide di usion h ough 431
he channels, o iginally p esen in he ma ix, and enla ged in he cou se o he lipase-media ed 432
lipids deg ada ion [78]. These indings sugges ha a selec ion o he lipidic componen s is 433
impo an in o de o modula e he p o ein/pep ide elease. 434
435
I is impo an o highligh he possibili y o an in e ac ion be ween he pep ide/p o ein and he 436
lipid componen s and hei deg ada ion p oduc s. Fo example, in a wo k in ended o encapsula e 437
leup olide ace a e in o SLN using he nanop ecipi a ion echnique, he use o a hyd ophobic ion 438
pai ing complex be ween leup olide and sodium s ea a e led o a conside able educ ion o he 439
bu s e ec (1 h bu s elease: 10 % s. 45 %). Following his ini ial as elease, he pep ide was 440
slowly eleased o up o 2 days [80]. In ano he example he sus ained elease o sCT (40 - 45 % in 441
6 h) om chi osan-coa ed SLN p oduced by he double emulsion-sol en e apo a ion me hod was 442
a ibu ed o he high a ini y o he posi i ely cha ged sCT o he nega i ely cha ged lipids 443
(leci hin and ipalmi in) [28]. 444
445
The inco po a ion o PEG in o he lipid ma ix has also been p oposed as a s a egy o modula e 446
he elease p o ile. Fo example, he elease o insulin om SLNs p oduced by he supe c i ical 447
luid echnology, could be con olled by inco po a ing 5 kDa PEG in he lipid mix u e as a po e-448
o ming agen [89]. Indeed, he o al amoun o insulin associa ed o PEG-con aining SLNs was 449
eleased in 3 days, whe eas PEG- ee SLNs needed 5 days o deli e hei con en . 450
451
F om he esul s in li e a u e up o da e (Table 3 shows some examples), we can conclude ha he 452
elease o pep ides om SLN is a ec ed by he composi ion o he lipidic ma ix (go e ning he 453
deg ada ion o he pa icles) and by he a ini y o he pep ide/p o ein owa ds he o mula ion 454
componen s. No mally, he in i o elease o he p o eins/pep ides is p olonged o a ew days, 455
howe e , i could be expec ed ha in an in i o si ua ion he p ocess could be accele a ed 456
depending on he deg ada ion a e o he lipidic ma ix. 457

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458
Table 3. Examples o pep ide/p o ein-loaded SLN ob ained h ough he di e en p epa a ion me hods: d ug loading and
459
elease p ope ies.
460
P epa a ion
me hod
Speci ic
s a egy
Pep ide/
P o ein AE (%) LC (%)
≤1h bu s
/ cumula i e
elease ( ime) – pH
medium
Re .
Mic o-
emulsion-
based
echnique
-
CyA
n.a.
6
-
13
<4 %
/ <4 % (2 h) pH 7.4
[
84]
Double
emulsion
[D-T p-6]
LHRH 90 n.a. <3 % / 10 % (8 h) pH 6.5 [10]
Thymo-
pen in 2 n.a. <5 % / 10 % (6 h) pH 6.5
[11]
Hyd ophobic
ion pai ing
Thymo
-
pen in 5 n.a. <5 % / 10 % (6 h) pH 6.5
Ho HPH - CyA 95 - 98
0.5
-
2
heo . n.a. [75]
96
1.9
n.a.
[90]
Cold HPH - CyA 79 - 94
0.5
-
2
heo . n.a. [75]
Lysozyme
43
-
59
0.03
n.a.
[76]
Emulsion –
sol en
e apo a ion
Double
emulsion sCT 31->90 n.a. <30 % / <45 % (6 h) pH 4
[28,
29]
Double
emulsion /
Re e se
micelliza ion
sCT 88 - 95 5 - 11
60
-
100 % / 100 % (2 h)
pH 6.8* [86]
Insulin 76-100 19
0
-
35 % / 60
-
90 % (6 d)
pH 7.4 [87]
Nanop eci-
pi a ion
-
Gonado
-
elin 50 - 69 n.a.
<30 % / <80 % (14 d)
pH 6.8 [79]
Leup o
-
lide 28 0.3
<45 % / 100 % (2 d)
pH 6.8 [80]
Hyd ophobic
ion pai ing
Leup o
-
lide 46 0.5
<10 % / 100 % (2 d)
pH 6.8
Supe c i ical
luid
echnology
- Insulin
20 - 80 1 - 4
0
-
17 % / 100 % (6 d)
pH 7.4 [89]
57 2.9
<10 %
/ 100 % (4 d)
pH 7.4 [91]
h-GH 48 2.4 <5 % / 100 % (4 d) pH 7.4
AE: associa ion e iciency (100 x associa ed pep ide mass / o al pep ide mass); CyA: cyclospo ine A; [D-T p-6] LHRH:
461
agonis ip o elin - lu einizing ho mone- eleasing ho mone; HPH: high p essu e homogeniza ion; LC: loading capaci y
462
(100 x pep ide mass / o al o mula ion mass); n.a.: no applicable; Re .: e e ences; h-GH: ecombinan human g ow h
463
ho mone; sCT: salmon calci onin; heo .: heo e ical; *Enzyme supplemen ed.
464
465
2.a.3. Mic oemulsions and Nanoemulsions
466
467
Bo h, wa e -in-oil (W/O) and oil-in-wa e (O/W) mic oemulsions a e usually conside ed as 468
he modynamically s able and iso opic sys ems, displaying sizes below 100 nm. The 469
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mic oemulsion o ma ion has been desc ibed as a spon aneous p ocess ha occu s a e mixing 470
he oil and he wa e phases con aining a ce ain amoun o su ac an s, in o de o achie e a low 471
in e acial ension be ween he wo phases [92]. Nanoemulsions ha e also been desc ibed as 472
colloidal dispe sions ha gene ally display sizes below 200 nm. In con as wi h mic oemulsions, 473
hese sys ems a e no iso opic. The nanoemulsion o ma ion equi es an ex e nal ene gy inpu in 474
o de o o e come hei posi i e ee ene gy and inc ease hei con ac a ea, leading o he 475
o ma ion o a kine ically s able colloidal dispe sion [93]. A special ype o emulsions is he one 476
p esen in he sel -emulsi ying d ug deli e y sys ems (SEDDS) and he sel -mic o-emulsi ying d ug 477
deli e y sys ems (SMEDDS) which ypically consis o mix u es o oil, su ac an and co-su ac an s. 478
Recen ly, many o hese SMEDDS ha e been classi ied as sel -nanoemulsi ying sys ems (SNEDDS) 479
[94]. Among he wide a ie y o lipids, he long and medium chain glyce ides and a y acids a e 480
he mos commonly used o he p epa a ion o sel -emulsi ying sys ems, mic oemulsions and 481
nanoemulsions con aining pep ides [25]. Medium chain a y acids a e known o imp o e he 482
pep ide solubili y and acili a e he emulsi ica ion p ocess since hei mix u e wi h he aqueous 483
phase is easie . 484
485
2.a.3.a. P epa a ion echniques 486
487
A wide a ie y o me hods ha e been de eloped o p oduce mic o/nanoemulsions. These 488
echniques can be classi ied depending on he p ocedu e used o supply ene gy o he sys em [95–489
97], being b oadly ca ego ized in o he ollowing wo g oups: i) High-ene gy p ocesses, which 490
imply he applica ion o mechanical and in ensi e dis up i e o ces o he di e en phases o he 491
sys em. Special de ices a e necessa y in o de o in e mingle he oily and he aqueous phases, 492
leading o he o ma ion o nanod ople s (homogeniza ion, mic o luidiza ion and ul asonica ion) 493
[98–100]; ii) Low-ene gy p ocesses (spon aneous emulsi ica ion and phase in e sion), which a e 494
based on he spon aneous o ma ion o nanoemulsions ei he by changing he composi ion (i.e., 495
a io su ac an :oil:wa e , addi ion o sal s, e c.) o he p ocess condi ions (i.e., empe a u e- ime 496
p o ile, s i ing, addi ion speed, e c.) [97,101–104]. Among he wide a ie y o echniques, hose 497
based on he spon aneous emulsi ica ion a e, so a , he mos commonly used o he associa ion 498
o pep ides/p o eins. This is mainly due o he ac ha his me hod a oids he pep ides/p o eins 499
being exposed o any empe a u e o p essu e s ess. 500
501
i) Spon aneous Emulsi ica ion. 502
Th ough his me hod, he nanoemulsion is spon aneously o med upon he mix u e o he oily and 503
he aqueous phases (Fig. 11) [105,106]. The p o ein/pep ide is included in one o hem depending 504
on i s hyd ophilici y o inco po a ed in o he oily phase in a small amoun o wa e . Bo h phases 505
a e immiscible in each o he ; howe e , one o he componen s p esen in one o hem (i.e., an 506
o ganic sol en , a su ac an ) is pa ially miscible in bo h. Once he wo phases a e in con ac , a 507
non-equilib ium s a e is o med, causing he apid shi ing o he miscible componen om i s 508
o iginal phase in o he o he . This ac will lead o an inc ease in he oil-wa e in e acial a ea and 509
u bulence, p omo ing he spon aneous o ma ion o he nanoemulsion [107]. 510
511
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512
Figu e 11. Schema ic iew o he spon aneous emulsi ica ion echnique o p oduce nanoemulsions
513
514
2.a.3.b. Cha ac e iza ion, pep ide/p o ein loading, ac i i y and elease p o ile 515
516
- Pa icle size dis ibu ion: by selec ing app op ia ely he ing edien s and he p epa a ion me hod, 517
emulsions showing a wide ange o sizes, cha ges and physical p ope ies can be ob ained. The 518
inal size dis ibu ion o he emulsion can be modula ed by op imizing i s composi ion 519
(concen a ion o he componen s, a io su ac an :oil:wa e , in e acial ension, iscosi y, 520
emulsi ie adso p ion kine ics, e c.) and he ope a ing condi ions ( empe a u e- ime p o ile, 521
s i ing a e, p essu e, ampli ude o sonica ion and numbe o cycles, e c.) [108–111]. The use o 522
e na y phase diag ams is an use ul ool o p edic he op imum condi ions o he o ma ion o 523
he nanoemulsion [112,113]. 524
525
- Pep ide/p o ein loading and ac i i y: he combina ion o he spon aneous emulsi ica ion 526
echnique wi h se e al speci ic s a egies, such as, double emulsi ica ion, e e se micelliza ion, 527
hyd ophobic ion pa ing o pep ide-lipid/su ac an in e ac ion (sec ion 2.a.) has been e ec i e o 528
he loading o hyd ophilic pep ides (Table 4) such as insulin, wi h AEs highe han 85 % 529
[27,35,114,115]. Among he ac o s in luencing his associa ion, i has been ound ha small 530
a ia ions in he inal pH ( om 6.5 o 6.8), may lead o sha p dec eases in he AEs om 79 o 30 %. 531
This esul was a ibu ed o he di e en ioniza ion deg ee o bo h, he pep ide and he polyme 532
a he selec ed pHs, and hei elec os a ic and/o hyd ophobic in e ac ions [116]. Despi e he 533
good associa ion e iciencies achie ed, he loading capaci y o hese sys ems is usually lowe han 534
1 % [31,117]. 535
536
A e sys em p epa a ion, he loaded pep ide/p o ein mus be able o keep i s ac i i y. In ac , 537
he e a e some ope a ion condi ions, i.e. he use o o ganic sol en s and su ac an s, which can 538
lead o p o ein dena u a ion and/o agg ega ion. High shea agi a ions, empe a u es o p essu es 539
can a ec he in eg i y o he p o ein, as well [31,118,119]. In his ega d, he use o ELISA assays 540
has been epo ed as an e icien me hod o unde s and i he ac i i y o he encapsula ed p o ein 541
is kep . Howe e , in he speci ic case o mic o- and nanoemulsions, di ec in i o e alua ion o he 542
o mula ion is he main app oach epo ed o e alua e he e icacy o he loaded he apeu ic 543
agen [118,120]. 544
545
- Pep ide/p o ein elease: only a ew pape s ha e been published dealing wi h he mechanism 546
behind he elease o he p o ein/pep ide d ugs om mic o/nanoemulsions. In gene al, he 547
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elease o he d ug has been ela ed o i s pa i ion be ween he emulsion and he su ounding 548
medium and also o he al e a ion/deg ada ion o he lipidic componen s. Fo example, when hey 549
a e o ally adminis a ed, hei con ac wi h he gas oin es inal luids can cause a phase in e sion 550
o sepa a ion o he emulsion phases, ha may lead o a p ema u e d ug elease [120,121]. The 551
con e sion o hese liquid sys ems in o solid o ms h ough eeze d ying, sp ay d ying, mel 552
g anula ion, mel ex usion o adso p ion o e solid ca ie s has been p oposed as a way o 553
o e come he colloidal ins abili y o hese sys ems [122]. Fu he imp o emen s o his echnology 554
in o de o op imize he deli e y o hyd ophilic d ugs om sel -emulsi ying sys ems a e s ill 555
needed. Howe e , o lipophilic pep ides, some o mula ions, such as Neo al® (SMEDDS con aining 556
cyclospo ine) ha e al eady been ma ke ed [1]. 557
558
Table 4. Examples o pep ide/p o ein-loaded mic o/nanoemulsions and SEDDS/SMEDDS/SNEDDS ob ained h ough he
559
spon aneous emulsi ica ion me hod: d ug loading and elease p ope ies.
560
Sys em Speci ic
s a egy
Pep ide/
P o ein AE (%) LC (%)
≤1h bu s / cumula i e
elease ( ime) – pH
medium
Re .
O/W -
sCT > 90 n.a. n.a.
[119,
123]
Pli i
-
depsin 95-98 0.54 n.a. [124]
W/O
-
TAT 97
0.006
TAMRA-
TAT
90 % (1 h) pH 6.8* [120]
hPTH1
-
34 83
n.a. (45
mg/mL)
100 % (50 min) pH 8*
65 % / 80 % (2 h) pH 2* [125]
Insulin +
ap o inin 97 0.1
(30
IU/g)
0 % (1 h) pH 1.2* [118]
Re e se
micelles Insulin > 85
n.a.
(2.2
% w/
heo .)
n.a. [27]
Hyd ophobic
ion pai ing Insulin
30
-
79
(complex-
a ion)
n.a. <10 % (1 h) pH 1.2* [116]
W/O/W Double
emulsion
Insulin 96 - 97 n.a.
(18
IU/g)
0
-
80 % / 0
-
80 % (1.5 h)
pH 7 [114]
Insulin +
ap o inin 88 - 97
0.075
20
-
30 % / 20
-
30 %
(2 h)
pH 7 [115]
sCT +
ap o inin n.a.
n.a.
(400
IU/g
heo .)
90 % / 100
% (
2
h
)
pH 6.4/1.2*
80 % / 80 % (2 h) pH 7.5*
[126]
SMEDDS
SNEDDS
Hyd ophobic
ion pai ing
Insulin
64
-
71
0.3
-
1.1
15 % / 30 % (8 h) pH 7.4
[117]
Leup o-
elin
59
(complex-
a ion)
0.4
heo .
complex
<20 % /
40 % o complex
(30 h) pH 6.8 [31]
Hyd ophilic
-
hyd ophobic Insulin 85 - 99 n.a. 1 % / 14 % (24 h) pH 7.4 [35]
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in e ac ions
AE: associa ion e iciency (100 x associa ed pep ide mass / o al pep ide mass); LC: loading capaci y (100 x pep ide mass
561
/ o al o mula ion mass); n.a.: no applicable; Re .: e e ences; hPTH1-34: ecombinan 1-34 N- e minal agmen o
562
endogenous human pa a hy oid ho mone; sCT: salmon calci onin; TAMRA: e ame hyl hodamine; TAT: HIV
563
ansac i a o o ansc ip ion; heo .: heo e ical; *Enzyme supplemen ed.
564
565
2.a.4. Nanocapsules
566
567
Nanocapsules a e co e-shell s uc u ed d ug deli e y ca ie s. They consis o an oily co e which is 568
s abilized by su ac an s and i is su ounded by one o mo e polyme shells [127]. Bo h, co e and 569
ou e shell laye s, play a c ucial ole in he ou come o he o mula ion: whe eas he co e usually 570
wo ks as a d ug ese oi , he polyme coa ing helps he associa ed d ug o o e come biological 571
ba ie s and modula e i s elease p o ile. Among he wide a ie y o lipids, he long chain a y 572
acids and he medium chain glyce ides (mono-, di- and i-), bo h showing pene a ion enhance 573
p ope ies, a e he mos commonly used o p oducing nanocapsules [25]. 574
575
2.a.4.a. P epa a ion echniques 576
577
The p epa a ion o nanocapsules in ol es he emulsi ica ion o an oily phase in o an aqueous 578
phase. The polyme o ming he shell can be inco po a ed in o he o ganic phase o he aqueous 579
phase [128,129]. Addi ionally, wo di e en polyme s can be inco po a ed one in each phase 580
[130,131]. The shell is o med due o i s p ecipi a ion a he in e phase o o an ionic in e ac ion 581
be ween he oily co e and he polyme . In a di e en si ua ion, i.e. poly(alkylcyanoac yla es), he 582
polyme shell is o med due o an in e acial polyme iza ion p ocess [9,132]. The main ac o s 583
d i ing he choice o he app op ia e nanocapsules p oduc ion echnique a e he na u e o he 584
polyme as well as ha o he pep ide/p o ein o be encapsula ed (Table 5). 585
586
Table 5. Main cha ac e is ics o he mos commonly used p epa a ion me hods o nanocapsules
587
Technique P inciple S ess Exposu e
O ganic
sol en s
Simpli
-
ci y
“In si u”
polyme iza ion
/ In e acial
polyme iza ion
Oily co e
nanocapsules
Monome s
polyme iza ion “in
si u” a he in e -
ace o an emulsion
Undesi able
eac ions d ug-
monome s /
Vigo ous s i ing
Yes
+
Aqueous co e
nanocapsules
No
necessa ily
Polyme
p ecipi a ion/
deposi ion
Sol en
displacemen
Sol en di usion o
he aqueous phase
and polyme p eci-
pi a ion/deposi ion
Mode a e
s i ing Yes ++
Sel -emulsi i-
ca ion
Su ac an shi ing
om he oily o he
aqueous phase and
polyme deposi ion
High su ac an
concen a ion /
Mode a e
s i ing
No ++
588
i) “In si u” polyme iza ion. 589

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In his me hod, which is also named as in e acial polyme iza ion, he polyme o ma ion occu s 590
“in si u” a he in e ace o an emulsion h ough a as polyme iza ion among eac i e monome s. 591
Due o hei apid and easy polyme iza ion, alkylcyanoac yla es ha e been he monome s o 592
choice o his pu pose [133,134]. Un o una ely, he po en ial eac ion be ween he d ug and he 593
eac i e monome s du ing he p ocess cons i u es a limi a ion o his app oach [135]. 594
595
- In e acial polyme iza ion in oily co e nanocapsules. In his case, he o ganic phase is composed 596
by he pep ide/p o ein, he oil, he monome s and an o ganic sol en . The sol en needs o be 597
wa e -miscible in o de o p omo e i s di usion owa ds he aqueous phase, allowing he 598
spon aneous o ma ion o nanome ic oily d ople s [136]. The o ganic phase is usually injec ed 599
in o he aqueous phase, which con ains a leas a hyd ophilic su ac an . This p ocess is usually 600
pe o med unde igo ous s i ing, leading o he ins an aneous o ma ion o he nanocapsules 601
(Fig. 12). An addi ional inal s ep o emo e he o ganic sol en s can be pe o med [9,134]. 602
603
604
Figu e 12. Schema ic iew o he in e acial polyme iza ion echnique o p oduce oily co e nanocapsules
605
606
- In e acial polyme iza ion in aqueous co e nanocapsules. In his me hod, he aqueous phase, 607
which con ains he p o ein/pep ide, wa e and some imes wa e -miscible sol en , is emulsi ied 608
in o an o ganic phase consis ing o an oil and a lipophilic su ac an using sonica ion o igo ous 609
s i ing. Once he W/O emulsion is o med, he monome s a e added unde mechanical s i ing. 610
This las s ep, igge s he polyme iza ion a he W/O in e ace and leads o a inal sys em 611
consis ing o aqueous co e nanocapsules dispe sed in oil (Fig. 13) [137,138]. The nanocapsules a e 612
inally isola ed by ul acen i uga ion ollowed by hei esuspension in wa e [139,140]. 613
614
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615
Figu e 13. Schema ic iew o he in e acial polyme iza ion echnique o p oduce aqueous co e nanocapsules
616
617
ii) Polyme p ecipi a ion/deposi ion 618
Con a ily o he “in si u” polyme iza ion, he use o p e o med polyme s allows a good con ol o 619
he inal polyme molecula weigh , a oiding undesi able eac ions be ween he d ug and 620
monome s. In his case, he polyme coa ing can be o med by ei he polyme p ecipi a ion o 621
polyme deposi ion/in e ac ion. 622
623
- Polyme p ecipi a ion. This echnology was i s epo ed by Fessi and cowo ke s [141,142]. This 624
me hod in ol es he use o an o ganic pola phase con aining a lipophilic su ac an , an oil, and 625
he polyme , and an aqueous phase, ha may con ain hyd ophilic su ac an s. The usual 626
p ocedu e can be summa ized as ollows (Fig. 14): he o ganic phase is added d opwise o e he 627
aqueous phase unde mode a e s i ing leading o he ins an aneous di usion o he wa e -628
miscible sol en om he lipophilic solu ion o he aqueous phase. As a consequence, he polyme 629
p ecipi a es a he in e ace o he o med oily d ople s, s abilizing hem. In a inal s ep, sol en s 630
can be emo ed by e apo a ion unde acuum [128,130,142]. 631
632
- Polyme deposi ion/in e ac ion. Al e na i ely, nanocapsules can be p oduced using wa e 633
soluble polyme s acco ding o a deposi ion/in e ac ion echnique. In his case, he polyme shell is 634
o med due o i s ionic in e ac ion wi h he lipophilic componen s o he oily co e. This in e ac ion 635
may occu du ing he sol en displacemen p ocess o a e he incuba ion o he p e o med 636
nanoemulsion wi h he wa e -soluble polyme [123,129,143,144]. Addi ionally, he possibili y o 637
ob aining mul i-laye nanocapsules has been epo ed. This laye by laye app oach is based on he 638
adso p ion o di e en polyme ic laye s on o a colloidal empla e. The addi ion o each polyme ic 639
laye should in e he o e all cha ge o he sys em in all he abso p ion s eps [127]. Ou g oup 640
has epo ed he possibili y o ob aining p o ein-loaded nanocapsules by igge ing he polyme 641
deposi ion by a sel -emulsi ica ion me hod a oiding he use o o ganic sol en s. The p inciple o 642
his echnique is he same desc ibed in sec ion 2.a.3.a. o he spon aneous o ma ion o 643
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mic o/nanoemulsions (Fig. 11), including, addi ionally, a wa e -soluble polyme in o he aqueous 644
phase [145] o in a subsequen incuba ion s ep [146,147]. 645
646
647
Figu e 14. Schema ic iew o he sol en displacemen - polyme p ecipi a ion/deposi ion echnique o p oduce
648
nanocapsules
649
650
2.a.4.b. Cha ac e iza ion, pep ide/p o ein loading, ac i i y and elease p o ile 651
652
- Pa icle size dis ibu ion: he main ac o s a ec ing he inal pa icle size dis ibu ion o 653
nanocapsules a e he a io and he mixing condi ions be ween he wo phases, as well as he 654
physicochemical p ope ies and concen a ion o he di e en componen s 655
[119,128,129,148,149]. O e all, nanocapsules ha e been p oduced so a wi h a size be ween 30 656
and 400 nm. 657
658
- Pep ide/p o ein loading and ac i i y: Cou eu and cowo ke s we e he i s epo ing he 659
possibili y o using nanocapsules as deli e y ehicles o p o eins [9]. Since hei con ibu ion 660
h ough he encapsula ion o insulin in poly(alkylcyanoac yla e) nanocapsules, se e al au ho s 661
ha e demons a ed he capabili y o nanocapsules o en ap di e en pep ides/p o eins (Table 6). 662
Despi e he high AEs a ained, he LC alues epo ed so a a e below 2 %, which is usually due o 663
he ha d solubiliza ion o hyd ophilic pep ides in o he lipidic phase and hei endency o di use 664
o he ou e aqueous phase [139]. When nanocapsules a e ob ained by in e acial polyme iza ion, 665
he monome concen a ion has been p o ed o be one o he main ac o s in luencing he 666
pep ide associa ion e iciency [150]. The pH o he pep ide solu ion has also been shown o 667
in luence he AE o pep ides o PACA nanocapsules. This e ec is a ibu ed o he in luence o he 668
pH on he polyme iza ion a e o he polyme [148]. 669
670
Ou g oup has also shown he possibili y o a ach p o eins o p e o med polyme nanocapsules. 671
Fo example, we ha e e icien ly associa ed he ecombinan hepa i is B su ace an igen ( HBsAg) 672
on o p e o med chi osan nanocapsules. In his si ua ion he a achmen o he p o ein was ound 673
o be dependen on bo h p o ein and nanocapsules concen a ion and he mechanism o 674
a achmen was based on ionic/hyd ophobic in e ac ions [151–153]. 675
676
Di e en o mula ion pa ame e s could in luence he pep ide/p o ein s uc u e. Wi h “in si u” 677
polyme iza ion he d ug could wo k as a monome du ing he polyme iza ion p ocedu e, being 678
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dena u ed and losing i s ac i i y. Howe e , e hanol can be used o p ese e he pep ide/p o ein 679
s uc u e [154]. Fu he mo e, in all he echniques desc ibed abo e o nanocapsules p oduc ion, 680
he p esence o o ganic sol en s and su ac an s, as well as he igo ous s i ing, could also a ec 681
he s uc u e o he encapsula ed pep ide/p o ein [139,143,148]. Elec opho esis-based 682
echniques (e.g. na i e SDS-PAGE), HPLC-based me hods o ci cula dich oism ha e been epo ed 683
o s udy he s uc u al s abili y o nanoencapsula ed pep ides/p o eins [139,148,154]. Howe e , in 684
he majo i y o he wo ks, he ac i i y o he encapsula ed d ug was e alua ed a e i s in i o 685
adminis a ion [128,143,155]. 686
687
- Pep ide/p o ein elease: he mechanism d i ing he elease o pep ides/p o eins en apped in o 688
nanocapsules has been de ined as a combina ion o wo main p ocesses: he pa i ion o he d ug 689
be ween he nanoca ie and he ex e nal elease medium and he deg ada ion o he polyme 690
shell and he lipid co e. Bo h p ocesses can be a ec ed by di e en ac o s, such as he pH o he 691
elease medium, he na u e o he lipidic co es, he ype and molecula weigh o he polyme , as 692
well as he hickness o he polyme shell [138,143,149,156]. BSA cumula i e eleases anging 693
om 35 % up o 90 % we e epo ed o poly(bu ylcyanoac yla e) nanocapsules a e 8 h in elease 694
media wi h di e en pHs ( om 2.5 o 8.5) and di e en p o iles we e showed when 695
poly(bu ylcyanoac yla e) o 4, 7 o 10 kDa was used. Likewise, he loading and he molecula 696
weigh con e he p o ein wi h di e en di usion capaci ies and speci ic in e ac ions wi h he 697
componen s o he sys em. High loadings inc ease he p o ein g adien be ween he nanocapsule 698
co e and he ou e phase, and p o eins wi h high molecula weigh s di use mo e slowly h ough 699
he polyme ic wall [139]. On he o he hand, when he p o ein is a ached o he polyme shell, 700
he mechanism o elease is based on i s disassocia ion [157] and his p ocess is no mally 701
dependen on he pH and ionic s eng h o he elease medium. 702
703
F om he esul s in li e a u e up o da e, we can conclude ha he sol en displacemen echnique 704
is he mos ad an ageous o encapsula ing hyd ophilic pep ides in nanocapsules. Apa om i s 705
simplici y, and he possibili y o con olling he exac molecula weigh o he polyme and 706
a oiding undesi able c oss- eac ions, high associa ion e iciencies can be a ained. 707
708
Table 6. Examples o pep ide/p o ein-loaded nanocapsules ob ained by he di e en p epa a ion me hods: d ug loading
709
and elease p ope ies.
710
P epa a ion
me hod
Pep ide
/
P o ein AE (%) LC (%)
≤1h bu s
/ cumula i e elease
( ime) – pH medium Re .
In e acial
polyme iza ion
(Oily / Aqueous
co e)
Insulin
55 - 98 n.a. n.a.
[9,158,
159]
90 n.a. (0.45
mg/mL)
10 % / 13 % (5 h) pH 7.4/1-2*
77 % / 80 % (5 h) pH 6-7*
[154,
160,
161]
57 - 95 n.a. n.a.
[148,
162]
100
n.a.
n.a.
[163]
Human
35
-
79
n.a. (0.0067
-
40
-
60 % (20 min) pH 7.4*
[164]
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mechanism o elease is mainly d i en by he deg ada ion o he polyme , he na u e o he 872
p o ein may also in luence i s solubili y, i s in e ac ion wi h he polyme and i s di usion ac oss 873
he channels gene a ed in he polyme deg ada ion p ocess [171,183,188,190,194]. 874
875
Table 8 shows examples o p o eins associa ed o PLGA nanopa icles p oduced by di e en 876
echniques. 877
878
Table 8.Examples o pep ide/p o ein-loaded PLGA nanopa icles ob ained h ough di e en p epa a ion me hods: d ug
879
loading and elease p ope ies.
880
P epa a ion
me hod
Pep ide/
P o ein AE (%) LC (%)
≤
1
h bu s
/
cumula i e
elease
( ime) - pH medium Re .
Double emulsion -
sol en
e apo a ion
BSA
70
-
80
0.7
-
0.8
n.a. / 80 % (
28
d
)
pH 7.4
[190]
Insulin
70
-
80
3.
5
-
4
n.a.
/ 20 % (
28
d
)
pH 7.4
Cyclospo ine
A
60
-
90
n.a.
15
-
25 % / 70
-
90
% (
24
h
)
pH 7.4
[195]
BSA
28
-
88
n.a.
n.a. / 40
-
100
%
(28 d) pH 7.4
[178]
HSA
22
-
33
1.3
-
2.6
n.a.
[196]
Te anus oxoid 31 - 37 n.a. n.a. / 7 - 18 % (1 d) pH 7.4
<7 / 4 - 15 % (4 h) pH 1.2*/7.5*
[170,
171]
L
-
Aspa a
ginase
15
-
40
1.8
-
4.9
n.a./
15
-
95 % (21 d) pH 7.4
[189]
Insulin
n.a.
n.a.
n.a. / 70 % (40 d) pH
n.a.
[197]
IGF
-
1
22
-
43
n.a.
n.a. / 78 % (40 d) pH
n.a.
Emulsion -
sol en di usion
BSA 4 - 60
1
-
4
heo .
60
-
80
%
/
80
-
90 % (
14
d
)
pH 7.4 [188]
IgG n.a.
1
-
4
heo . 5 - 25 % / 10-30 % (14 d) pH 7.4
Insulin
20
-
40
0.2
-
0.4
20 % / 80 % (
14
h
)
pH 7.4
[181]
PDGF
-
BB
87
0.01
40 % / 80 % (
40
d
) pH 7.4
[182]
FGF
-
2
68
0.01
40 % / 80 % (
40
d
) pH 7.
4
Nanop ecipi a ion
Insulin
14
-
23
0.3
-
0.5
n.a.
[184]
Lysozyme
35
-
91
0.7
-
1.8
n.a.
α-chymo ypsin
11 - 71
2
-
5
heo . n. a. [185]
Cy
-
c
72
3.6
n. a. / 100 % (120 d)
pH 7.3
AE: associa ion e iciency (100 x associa ed pep ide mass / o al pep ide mass); BSA: bo ine se um albumin; Cy -c: ho se
881
hea cy och ome c; FGF-2: ib oblas g ow h ac o ; HAS: human se um albumin; IGF-1: insulin-like g ow h ac o ; IgG:
882
immunoglobulin G; LC: loading capaci y (100 x pep ide mass / o al o mula ion mass); n.a.: no applicable; PDGF-BB:
883
pla ele -de i ed g ow h ac o ; Re .: e e ences; heo .: heo e ical; *Enzyme supplemen ed.
884
885
2.b.2. Ac ylic polyme s-based nanopa icles
886
887
Following he pionee ing wo k o P. Speise and co-wo ke s on he associa ion o an igens (human 888
immunoglobulin G and e anus oxoid) o polyac ylamide nanopa icles in 1976 [8], di e en ypes 889
o ac ylic polyme s ha e been used o p oduce nanopa icles, including polyac ylic acid, 890
polyac ylamides, polyme hylme hac yla es and poly(alkylcyanoac yla es) [198]. These syn he ic 891
polyme s a e conside ed o be biocompa ible and, in some cases, biodeg adable polyme s 892

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[199,200]. Among hem, poly(alkylcyanoac yla es) (PACA) a e he mos commonly used o 893
p epa ing nanopa icula e sys ems and, in pa icula , o he deli e y o p o eins. Thei ni ile and 894
es e g oups a e elec on a ac i e unc ional g oups and his p ope y makes he inyl ca bon o 895
he monome eally eac i e, hence, able o polyme ize in he p esence o an ini ia o . F ee 896
adical, anionic o zwi e ionic polyme iza ion a e he main app oaches adop ed so a o he 897
p oduc ion o PACA nanopa icles [200–202]. O e all, despi e he ea ly de elopmen and 898
a en ion ha hese pa icles ecei ed in he pas , only a ew pape s desc ibing hei use o 899
p o ein deli e y ha e been ound in he li e a u e. 900
901
2.b.2.a. P epa a ion echniques 902
903
Apa om he in e acial polyme iza ion me hod, which has been mainly used o oily co e 904
nanocapsules p oduc ion, and as such, i was desc ibed in he p e ious sec ion (Sec ion 2.a.4.a.), 905
wo main s a egies (summa ized in Table 9) ha e been desc ibed o syn hesize polyac yla e-based 906
nanos uc u es: he anionic polyme iza ion and he ee adical dispe sion polyme iza ion 907
echniques. In bo h cases, he use o ganic sol en s is a oided, being he main sou ce o p o ein 908
ins abili y i s po en ial eac i i y wi h he monome . 909
910
Table 9. Main cha ac e is ics o he mos commonly used echniques o o m pep ide/p o ein-loaded polyac yla e-based
911
nanopa icles
912
Technique P inciple S ess exposu e O ganic
sol en s Simplici y
Anionic
polyme iza ion
Monome s
polyme iza ion due o
OH
-
g oups in he
medium
Undesi able eac ions
d ug-monome s No +
F ee adical
dispe sion
polyme iza ion
Monome s
polyme iza ion due o
he gene a ion o ee
adicals and c osslinking
Undesi able eac ions
d ug-monome s-
c osslinking agen / F ee
adicals / UV / Hea
No +
913
i) Anionic polyme iza ion 914
In his echnique, he ac ylic monome s, a s abilize and an ini ia o (OH
-
in wa e ) a e necessa y o 915
o m he nanopa icles. The monome s, which a e poo ly soluble in wa e , a e emulsi ied in o an 916
acidic wa e solu ion (pH 2 - 4) con aining he s abilize ( ypically dex an). Once he d ople s a e 917
o med, he monome s a s o polyme ize hanks o he hyd oxyl ions (OH
-
) p esen in he wa e 918
phase (Fig.19). The acidic pH slows down he polyme iza ion a e, he eby con olling he p ocess 919
o pa icles o ma ion [133,201]. P o eins can be a ached on o he su ace o he pa icles, o 920
simply inco po a ed in o he eac ion mix u e du ing pa icles o ma ion [203–207]. 921
922
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923
Figu e 19. Schema ic ep esen a ion o he anionic-polyme iza ion echnique o p oduce polyac yla e-based
924
nanopa icles
925
926
ii) F ee adical dispe sion polyme iza ion. 927
Peppas and co-wo ke s used his echnique o ob ain gel nanosphe es h ough a pho o- o 928
he mal-ini ia ed polyme iza ion (Fig. 20). This echnology in ol es he use o speci ic ini ia o s as 929
well as a c osslinking agen . The monome s (i.e. me hac ylic acid, MAA and monome hyle he 930
monome hac yla e, PEGMA), he c osslinking agen (i.e. e a (e hylene glycol) dime hac yla e) 931
and he ini ia o (i.e., 1-Hyd oxylcyclohexyl phenyl ke one) a e solubilized in an aqueous phase. 932
Once he ini ia o is ac i a ed (UV, hea ), he o ma ion o oligome s and c osslinks s a s. Finally, 933
since he polyme is no soluble in wa e , nuclei o polyme iza ion a e c ea ed leading o he 934
o ma ion o nanosphe es (i.e. P(MAA-g-PEG)). Once he polyme iza ion is comple ed, 935
nanosphe es a e pu i ied by epea ed washing s eps o emo e he un eac ed monome s and he 936
associa ion o he p o ein (i.e. insulin, OVA) is ca ied ou in a subsequen incuba ion s ep 937
[208,209]. 938
939
940
Figu e 20. Schema ic ep esen a ion o he ee adical dispe sion polyme iza ion echnique o p oduce polyac yla e-
941
based gel nanosphe es
942
943
2.b.2.b. Cha ac e iza ion, pep ide/p o ein loading, ac i i y and elease p o ile 944
945
- Pa icle size dis ibu ion: in gene al, polyac yla e-based nanopa icles desc ibed in he li e a u e 946
ha e a size in he ange o 50 nm and 500 nm and a nega i e su ace cha ge [210–212]. Di e en 947
pa ame e s can a ec he polyme iza ion p ocess and, as a consequence, he physicochemical 948
p ope ies o PACA nanopa icles. The mos impo an pa ame e , which allows he con ol o he 949
polyme iza ion a e and, hence he pa icle o ma ion is he pH, howe e , he monome 950
concen a ion also has a signi ican in luence in his p ocess. Finally, he empe a u e and he 951
addi ion o su ac an s ha e also been desc ibed as a way o modula e he pa icle size [203,212–952
215]. 953
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954
- Pep ide/p o ein loading and ac i i y: Table 10 gi es an o e iew o he p ope ies o some 955
p o ein/pep ide-loaded polyac yla e-based nanopa icles o mula ions. The AE and LC alues 956
desc ibed in he li e a u e a e e y a iable, anging be ween 3.5 and 95 % AE and up o 26 % LC 957
[205,206,216]. Among he ac o s in luencing he AE, he ime a which he p o ein is added du ing 958
he polyme iza ion p ocess has been ound o be c i ical. Fo example, bo h insulin and GRF 959
(g ow h ho mone eleasing ac o ) eached a ound 85 % AE when hey we e added o he 960
polyme iza ion medium 30 minu es a e he p ocess s a ed [135,205]. 961
962
As o he “in si u” polyme iza ion me hod, he pep ide/p o ein could undesi ably wo k as a 963
monome du ing he polyme iza ion p ocedu e, which may esul in i s inac i a ion [206,217]. 964
Apa om echniques like HPLC o enzyma ic assays [203,217], di ec in i o e icacy o he 965
o mula ion has o en been used o es he in eg i y and ac i i y o he loaded pep ides/p o eins 966
[209]. 967
968
- Pep ide/p o ein elease: he elease o p o eins om polyac yla e-based nanopa icles is mainly 969
due o he bioe osion o he polyme ic ma ix [135]. Typically, hese pa icles show an ini ial bu s 970
elease, which can be bu e ed using addi i es. The p esence o dex an in o he o mula ion 971
medium could, o example, delay he elease o BSA om poly(α-bu ylcyanoac yla e) 972
nanopa icles [206]. P o ein elease has also been shown o be s ongly in luenced by he ype o 973
PACA used. Fo example, he elease o GRF was as e in he case o poly(isobu ylcyanoac yla e), 974
as compa ed o he case o poly(isohexylcyanoac yla e) nanopa icles. This was due o he 975
di e en bioe osion a es o he wo polyme s [135]. In he pa icula case o he polyac yla e-976
based gel nanosphe es (ac ylic acid (AA) o me hac ylic acid (MAA), hey we e speci ically 977
designed o exhibi a pH-dependen swelling and, hence, elease beha io [209]. This con ol could 978
be achie ed by adjus ing he polyme iza ion and c osslinking condi ions. 979
980
Table 10. Examples o pep ide/p o ein-loaded polyac yla e-based nanopa icles p epa ed by anionic and ee adical
981
dispe sion polyme iza ion: d ug loading and elease p ope ies.
982
P epa a ion
me hod
Pep ide/
P o ein AE (%) LC (%)
≤
1h bu s
/
cumula i e elease ( im
e)
-
pH
medium Re .
Anionic
polyme iza ion
Insulin
87
n.a.
n.a.
[205]
BSA
3.5
n.a.
15
-
55 %
/
70
-
90
% (
14
d
)
pH 7.4
[206]
SOD
7
-
33
n.a.
n.a.
[203]
NR1
6
-
10
n.a.
n.a.
GRF
80
n.a
70
% /
80
-
90
% (
8
h
) pH 7.4*
[135]
F ee adical
dispe sion
polyme iza ion
Insulin
65
2.1
n.a.
[208]
93
-
95
7
10
-
80 % / 100 % (3 h) 1h pH 3 + 2 h pH 7
[209]
OVA
51
26
0 % / 90
-
100 % (3 h) 1.5 h pH 3 + 2 h pH 7.4
[216]
AE: associa ion e iciency (100 x associa ed pep ide mass / o al pep ide mass); BSA: bo ine se um albumin; GRF: g ow h
983
ho mone eleasing ac o ; LC: loading capaci y (100 x pep ide mass / o al o mula ion mass); n.a.: no applicable; NR1:
984
an i-glu ama e N-me hyl D-aspa a e ecep o 1 an ibody; OVA: o albumin; Re .: e e ences; SOD: supe oxide
985
dismu ase; *Enzyme supplemen ed.
986
987
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2.b.3. Polysaccha ide-based nanopa icles
988
989
The mos commonly employed polysaccha ides o p o ein deli e y pu poses a e chi osan, 990
algina e, dex an and hyalu onic acid. Chi osan, a deace yla ed o m o chi in, is o med by 991
epea ed uni s o D-glucosamine and N-ace ylglucosamine [41,42,43]. Algina e is a block co-992
polyme made by α-gulu onic acid (pKa 3.4) and β-D-mannu onic acid (pKa 3.6) esidues linea ly 993
linked [220]. Like chi osan, i can be chemically modi ied on he acidic unc ional g oups o ob ain 994
he desi ed p ope ies [221,222]. Dex an is made by α (1→6) glucopy anoside uni s [223–225]. 995
The hyd oxyl g oups a e he main si es used o chemical modi ica ions, wi h dex an sul a e as 996
he mos common modi ied o m o d ug deli e y applica ions [226–228]. Finally, hyalu onic acid 997
is a linea polysaccha ide made by epea ed uni s o he disaccha ide o med by N-ace yl D–998
glucosamine and D–glucu onic acid [229]. These na u al polysaccha ides ha e in common he 999
p ope y o being wa e -soluble; howe e hei dis inc chemis y esul s in di e en pKa and 1000
unc ionali y in e ms o hei po en ial in e ac ion wi h di e en a ge s and hei capaci y o be 1001
modi ied wi h di e en ligands. Among he polysaccha ide-based nanopa icles desc ibed so a , 1002
hose made o chi osan we e o iginally de eloped in ou lab o he associa ion o p o eins 1003
[15,230]. Since his disco e y un il now, chi osan nanopa icles ha e been classi ied as he 1004
polyme ic deli e y nanopa icles ha ha e ecei ed he g ea es deal o a en ion. O e all, an 1005
ad an age o he echniques o he p oduc ion o polysaccha ide nanopa icles elies in he 1006
mildness o he p ocedu es [231–233], wi h he excep ion o he chemical c osslinking [234], 1007
which may lead o he dena u a ion o he p o ein. 1008
1009
Di e en echniques ha e been desc ibed un il now o p oduce polysaccha ide-based 1010
nanopa icles and nanocomplexes, being he mos commonly employed he ionic gela ion and he 1011
polyelec oly e complexa ion. Gene al speci ica ions o he di e en p epa a ion echniques a e 1012
p esen ed in Table 11. 1013
1014
Table 11. Cha ac e is ics o he mos commonly used echniques o o m polysaccha ide-based nanopa icles con aining
1015
pep ides/p o eins
1016
Technique P inciple S ess exposu e O ganic
sol en s Simplici y
Ionic
gela ion/c osslinking
Gela ion o he pa icles by
ionic c osslinking
I
onic in e a
c
ions
wi h he p o ein /
C osslinking agen
No + +
Polyelec oly e
complexa ion
Ionic in e ac ion be ween
polyme s o opposi e cha ge
Ionic in e ac ions
wi h he p o ein No + +
1017
i) Ionic gela ion/Ionic c osslinking 1018
Ou lab pionee ed he de elopmen o chi osan nanopa icles using he ionic gela ion/ionic 1019
c osslinking echnique [15,230], which has been la e ex ended o o he polysaccha ides such as 1020
algina e and dex an [235,236]. This echnique is based on he ac ha some cha ged 1021
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polysaccha ides can gel in aqueous solu ion in he p esence o small ions and c osslinking agen s 1022
(Fig.21) [133,222,237]. The ype o gelling agen is di e en based on he ype o polysaccha ide. 1023
Fo example, in he case o chi osan, ipolyphospha e (TPP) is he mos commonly c osslinking 1024
agen employed, while in he case o algina es, he use o calcium sal s (calcium chlo ide, calcium 1025
sul a e, o calcium ca bona e) is he mos common gela ion app oach [15,222,236,238–241]. 1026
1027
Al e na i ely, nanopa icles can be p oduced using a chemical c oss-linking eac ion. Howe e , his 1028
echnique has no been almos explo ed o he associa ion o p o eins [234] due o he po en ial 1029
chemical eac ions wi h he loaded p o ein. 1030
1031
1032
Figu e 21. Schema ic iew o he ionic gela ion/c osslinking echnique o p oduce polysaccha ide-based nanopa icles
1033
1034
ii) Polyelec oly e complexa ion 1035
Polyelec oly es complexes (PECs) a e complexes esul ing om he mixing o wo opposi ely 1036
cha ged mac omolecules (i.e., polyelec oly es). A schema ic ep esen a ion o he p ocedu e is 1037
shown in Figu e 22 [242,243]. The densi y o he cha ges and he cha ge dis ibu ion o e he 1038
polyme ic chains, in addi ion o he concen a ion o he wo polyelec oly es a e he main 1039
pa ame e s in luencing he p ope ies o he pa icles o med. The con ol o he ionic s eng h 1040
and pH o he eac ion medium, which in luences he deg ee o ioniza ion, is also undamen al o 1041
he nanopa icles o ma ion [244]. 1042
1043
1044
Figu e 22. Schema ic iew o he polyelec oly e complexa ion echnique o p oduce polysaccha ide-based nanopa icles.
1045
1046
2.b.3.b. Cha ac e iza ion, pep ide/p o ein loading, ac i i y and elease p o ile 1047
1048
- Pa icle size dis ibu ion: he ionic gela ion/c osslinking is, among he echniques desc ibed 1049
abo e, p obably he one allowing a be e con ol o he size. Indeed, in a epo by ou g oup 1050
[245], in ended o compa e he ionic c osslinking s. he ionic complexa ion o chi osan and pDNA, 1051
we showed ha he nanopa icles p epa ed by c osslinking o chi osan wi h TPP had a mo e 1052
con ollable size and a lowe polidispe si y han hose p oduced by ionic complexa ion. This esul 1053

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was a ibu ed o he ac ha he c osslinking wi h TPP led o he o ma ion o nanogelled 1054
pa icles wi h a ound and mo e de ined s uc u e [246,247]. O e all, he main ac o s in luencing 1055
he pa icle size dis ibu ion a e he a io and he concen a ion o he ionically in e ac ing species 1056
[15,227]. 1057
1058
- Pep ide/p o ein loading and ac i i y: in gene al, pa icles p oduced by gela ion o complexa ion 1059
a e cha ac e ized by a high LC, which can each alues up o 50 % and AE alues close o 100 % 1060
[15,227,247,248]. The p o ein associa ion e iciency is mainly a ec ed by he numbe o 1061
in e ac ing species and hei deg ee o ioniza ion. Fo example he AE o insulin o chi osan 1062
nanopa icles eached alues close o 90 %, howe e he alue dec eased o 37 % in he case o 1063
chi osan/glucomannan polyelec oly e complexes [247]. This was a ibu ed o he di e en pHs o 1064
he p o ein solu ion and also o a compe i ion be ween he p o ein and glucomannan o he 1065
chi osan posi i e si es. A simila compe i ion phenomenon was obse ed o he basic pep ide 1066
salmon calci onin, which was ound o compe e wi h p o amine in i s associa ion o hyalu onic 1067
acid/p o amine nanopa icles [249]. These a ini y/ionic compe i ion phenomena ha e been aken 1068
in o accoun o he modula ion o he LC. Fo example, he associa ion e iciency o insulin o 1069
chi osan-based nanopa icles could be inc eased om 66 % o 94 % when he anionic in e ac ing 1070
polyme s we e algina e and dex an sul a e espec i ely. This beha iou was explained due o he 1071
s ong ionic in e ac ions be ween he insulin and he sul a e g oups o dex an [250]. 1072
1073
The main sou ce o ins abili y o he loaded pep ide/p o ein is, in bo h ionic gela ion and 1074
polyelec oly e complexa ion, he possible ionic in e ac ion be ween he pep ide/p o ein and he 1075
polyme s/c osslinking agen s, which could d i e o p o ein dena u a ion [248,251,252]. 1076
Addi ionally, he acidic pH o en necessa y o p oduce nanopa icles by ionic gela ion (e.g. 1077
chi osan nanopa icles) can des abilize o a ec he pep ide/p o ein ac i i y (e.g. pH op imum o 1078
enzymes) [253]. Bo h elec opho esis-based echniques (i.e. SDS-PAGE and Wes e n blo ) and 1079
ELISA assays ha e been used o check i he pep ide/p o ein in eg i y and ac i i y we e p ese ed 1080
once included in polysaccha ide-based nanopa icles[251,252,254]. Likewise, spec oscopy-based 1081
echniques like FTIR ha e been used o s udy he in e ac ions be ween he unc ional g oups o 1082
he pep ide/p o ein and he polyelec oly es [255]. In he case o enzymes, he ac i i y was simply 1083
e alua ed h ough enzyma ic ac i i y assays [256]. Finally, in some cases, he ac i i y was only 1084
assessed a e hei in i o adminis a ion [248,257]. 1085
1086
- Pep ide/p o ein elease: om he poin o iew o d ug elease, nanopa icles p oduced by ionic 1087
gela ion o complexa ion no mally show an ionic s eng h-dependen elease p o ile, wi h an 1088
ini ial bu s elease. In ac , he sensi i i y o hese sys ems o pH changes and o he p esence o 1089
ions, is one o hei main d awbacks [228,247]. An example o his beha io has been obse ed o 1090
insulin-loaded dex an sul a e/polye hylenimine (PEI) nanopa icles p oduced by complexa ion, 1091
which comple ely eleased he pep ide in PBS 50 mM a e 5 minu es, while jus he 65 % o he 1092
pep ide was eleased in PBS 5 mM [228]. 1093
1094
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Among he o mula ion ac o s ha can be modi ied in o de o ha e a ce ain con ol o he 1095
elease p ocess, he combina ion o di e en coun e ac ing polyme s and su ac an s can be 1096
highligh ed. Fo example, we ha e shown ha he elease o BSA om chi osan nanopa icles 1097
p oduced by ionic c osslinking was a ec ed by he p esence o poloxame 188 in he o mula ion 1098
[15,230]. Simila ly, Sa men o e al compa ed he insulin elease p o ile om algina e/chi osan and 1099
dex an/chi osan nanopa icles [250]. They showed ha he elease o insulin was s ongly 1100
in luenced by ype o polyme s used, being he in e ac ion be ween he p o ein d ug and he 1101
polyme s undamen al o con ol he elease. These chi osan/algina e nanopa icles we e shown 1102
o ha e a pH-dependen elease p o ile, sui able o he gas ic and in es inal en i onmen . In ac , 1103
hese sys ems we e able o e ain he p o ein a he low pH o he s omach, and elease i in he 1104
in es ine, when he pH inc eased [236,258]. Swelling, dissocia ion, di usion and e osion a e 1105
epo ed as he main mechanisms behind p o ein elease om he nanopa icles made by ionic 1106
gela ion o polyelec oly e complexa ion [227,259]. 1107
1108
O e all, i could be concluded ha polysaccha ide-based nanopa icles a e hose leading o he 1109
highes p o ein loading capaci y, among hose indica ed in his e iew. The challenge ha emains 1110
associa ed o hese nanopa icles is ela ed o hei limi ed capaci y o con ol he elease in 1111
di e en physiologically ele an media. Ne e heless, he combina ion o di e en bioma e ials 1112
and su ac an s a e now seen as app oaches o o e come his hu dle. 1113
1114
Table 12 epo s examples o pep ides and p o eins encapsula ed in o polysaccha ide-based 1115
nanopa icles syn hesized by di e en s a egies. 1116
1117
Table 12. Examples o pep ide/p o ein-loaded polysaccha ide-based nanopa icles p epa ed by he di e en me hods:
1118
d ug loading and elease p ope ies
1119
P epa a ion
me hod
Pep ide
/
P o ein AE (%) LC (%)
≤
1
h bu s
/
cumula i e
elease
( ime) - pH medium Re .
Ionic gela ion/
c osslinking
Insulin
87 - 97 19 - 55
100
%
/
100 % (
2
h
)
pH 4/7
80 - 100 % / 100 % (2 h) pH 6.4 [248]
40 - 90
20
-
22
heo . 15 - 90 % / 15 - 90 % (2 h) pH 7.4
[247]
Immuno
-
modula o y
p o ein P1
10 - 30 16 - 21
heo . 10 -75 % / 10 - 75 % (2 h) pH 7.4
BSA
5
-
80
1
0
-
50
n.a. / 30
-
100
%
(
8
d
) pH
7
[15]
Te anus
Toxoid 50 10 n.a. [246]
VEGF
32
-
94
0.04
-
0.34
80
% /
>
90
%
(
24
h
) pH 7
[254]
PDGF
27
-
54
0.05
-
0.1
n.a. / > 90 % (
7
d
)
pH 7
Insulin 69 10
95 % / 95 % (2 h) pH 1.2
80 % / 80 % (2 h) pH 6.8 [250]
Polyelec oly e
complexa ion
BSA
70
n.a.
40
-
60 %
/ 40
-
60 % (7 h) pH 7.4
[255]
Insulin 66 - 94 5 - 13
55
-
100 % / 55
-
100 % (2 h) pH 1.2
70 - 100 % / 70 - 100 % (2 h) pH 6.8 [250]
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HBsAg
90
-
95
2.5
-
5
n.a.
[252]
sCT
100
10
-
39
55 % /
70
-
80
%
(
24
h
)
pH 7.4
[249]
TRIAL
n.a.
n.a.
n.a.
[257]
ARH pep ide
36
-
72
11
-
13
n.a. / 15
-
60 % (6
d) pH 7.4
[260]
OVA
80
-
85
7
-
38
n.a.
[220]
AE: associa ion e iciency (100 x associa ed pep ide mass / o al pep ide mass); BSA: bo ine se um albumin; INF-α:
1120
in e e on alpha; LC: loading capaci y (100 x pep ide mass / o al o mula ion mass); n.a.: no applicable; OVA:
1121
O albumin; PDGF: pla ele -de i ed g ow h ac o ; Re .: e e ences; HBsAg: ecombinan hepa i is B su ace an igen;
1122
sCT: salmon calci onin; heo .: heo e ical; TRIAL: umo nec osis ac o - ela ed apop osis inducing ligand; VEGF:
1123
Vascula endo helial g ow h ac o .
1124
1125
2.b.4. P o ein-based nanopa icles
1126
P o ein nanopa icles ha e been p oposed o a long ime as d ug deli e y sys ems due o hei 1127
low cos , easy p oduc ion, low cy o oxici y and biodeg adabili y [261,262]. A p o ein nanopa icle-1128
based p oduc o he deli e y o pacli axel (Ab axane®) has been app o ed by FDA and EMA, 1129
gene a ing a high in e es a ound his kind o pa icles. Recen wo ks ela ed o p o ein 1130
nanopa icles o p o ein deli e y ha e been epo ed in li e a u e, using gela in, HSA, BSA, g een 1131
luo escen p o ein (GFP) and silk ib oin as s a ing ma e ials o p oduce he pa icles [261]. 1132
1133
2.b.3.a. P epa a ion echniques 1134
1135
The p epa a ion me hod mos commonly used o p oduce p o ein nanopa icles is he desol a ion 1136
echnique, desc ibed below. 1137
1138
i) Desol a ion 1139
An aqueous solu ion o bo h he he apeu ic p o ein and he one used as a s a ing ma e ial o 1140
p oduce he pa icles is p epa ed. A desol a ing agen , like ace one, e hanol o dime hyl sul oxide 1141
(DMSO), is hen slowly added o he p o eins solu ion. A e he desol a ion p ocess, 1142
nanoagg ega es o he p o eins a e o med and a c osslinking agen , usually glu a aldehyde, is 1143
added, causing he o ma ion o s able pa icles (Fig.23) [262,263]. Al e na i ely o he chemical 1144
c osslinking, a coa ing wi h an ionic polyme (e.g., PEI) can be done o imp o e he s abili y o he 1145
pa icles [264]. 1146
1147
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1148
Figu e 23. Schema ic iew o he desol a ion echnique o p oduce p o ein nanopa icles
1149
1150
2.b.3.b. Cha ac e iza ion, pep ide/p o ein loading, ac i i y, and elease p o ile 1151
1152
- Pa icle size dis ibu ion: he size o he p o ein-based nanopa icles, which usually anges 1153
be ween 150 and 400 nm, depends on pa ame e s like he ype o c osslinke and he c osslinking 1154
ime. Thei su ace cha ge depends on he pH o he media and he ype o p o ein used o 1155
p oduce he pa icles [263–266]. 1156
1157
- Pep ide/p o ein loading and ac i i y: al hough he numbe o e e ences desc ibing he use o 1158
p o ein nanopa icles o p o ein deli e y is e y low, in gene al high AE alues a e epo ed in 1159
li e a u e (Table 13). Fu he mo e, he p esence o a polyme coa ing ha helps o e ain he 1160
p o ein d ug can also enhance he AE alues o p o ein nanopa icles, as demons a ed o 1161
albumin nanopa icles p epa ed by desol a ion wi h PEI o ming he polyme coa ing [264]. 1162
1163
The main d awback o he desol a ion p ocess is he use o o ganic sol en s o c osslinking agen s, 1164
which could dena u a e he pep ide/p o ein s uc u e, leading o p o ein inac i a ion. In his 1165
ega d, ELISA and enzyma ic assays ha e been used o check i he pep ide/p o ein ac i i y was 1166
e ained a e he nanopa icle o ma ion [262,264,265]. 1167
1168
- Pep ide/p o ein elease: a i s bu s elease ollowed by a sus ained elease p o ile is usually 1169
obse ed. The sus ained elease phase is associa ed o he deg ada ion and dissolu ion o he 1170
p o ein ma ix. The e o e, he elease is highly dependen on he ype o p o ein o ming he 1171
ma ix and also on i s in e ac ion wi h he p o ein ca go [264]. In he case o he PEI-coa ed BSA 1172
nanopa icles de eloped by Zhang and co-wo ke s, i was obse ed ha a he laye o PEI could 1173
educe he undesi ed elease o he p o ein d ug (bone mo phogene ic p o ein-2, BMP-2) om 70 1174
% o 15 % in he i s hou [264]. 1175
1176
Table 13. Examples o pep ide/p o ein-loaded p o ein-based nanopa icles: d ug loading and elease p ope ies
1177
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