Nanopharmaceuticals for eye administration: sterilization, depyrogenation and clinical applications

biology Review Nanopharmaceuticals for Eye Administration: Sterilization, Depyrogenation and Clinical Applications Aleksandra Zieli´nska 1,2 , Beatriz B. Soles 1, Ana R. Lopes 1, Beatriz F. Vaz 1, Camila M. Rodrigues 1, Thais F. R. Alves 3, Dorota Klensporf-Pawlik 4, Alessandra Durazzo 5, Massimo Lucarini 5, Patricia Severino 6,7,8,9 , Antonello Santini 10,* , Marco V. Chaud 3,* and Eliana B. Souto 1,11,* 1Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal; [email protected] (A.Z.); [email protected] (B.B.S.); [email protected] (A.R.L.); [email protected] (B.F.V.); [email protected] (C.M.R.) 2Institute of Human Genetics, Polish Academy of Sciences, Strzeszy´nska 32, 60-479 Pozna´n, Poland 3Laboratory of Biomaterial and Nanotechnology (LaBNUS). University of Sorocaba, Raposo Tavares 92.5, Sorocaba, 18078-005 São Paulo, Brazil; [email protected] 4Institute of Quality Science, Pozna´n University of Economics and Business, al. Niepodległo´sci 10, 61-875 Pozna´n, Poland; [email protected] 5CREA-Research Centre for Food and Nutrition, Via Ardeatina 546, 00178 Rome, Italy; [email protected].it (A.D.); [email protected].it (M.L.) 6Center for Biomedical Engineering, Department of Medicine, Brigham and Women& Hospital, Harvard Medical School, 65 Landsdowne Street, Cambridge, MA 02139, USA; [email protected] 7Biotechnological Postgraduate Program, University of Tiradentes (Unit), Av. Murilo Dantas, 300, 49010-390 Aracaju, Brazil 8Institute of Technology and Research (ITP), Nanomedicine and Nanotechnology Laboratory (LNMed), Av. Murilo Dantas, 300, 49010-390 Aracaju, Brazil 9Tiradentes Institute, 150 Mt Vernon St, Dorchester, MA 02125, USA 10 Department of Pharmacy, University of Napoli Federico II, 80131 Napoli, Italy 11 CEB—Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal *Correspondence: [email protected] (A.S.); mar[email protected] (M.V.C.); [email protected] (E.B.S.); Tel.: +39-81-253-9317 (A.S.); +55-15-98172-4431 (M.V.C.); +351-239-488-400 (E.B.S.) Received: 11 August 2020; Accepted: 13 October 2020; Published: 14 October 2020   Simple Summary: Nanopharmaceuticals have revolutionized the way ophthalmic drugs are administered to overcome ocular delivery barriers and improve drug bioavailability. The design and productionofanefficientoculardrugdeliverysystemstillremainachallenge. Inthisreview,wediscuss the sterilization and depyrogenation methods, commonly used for ophthalmic nanopharmaceuticals, and their clinical applications. Abstract: As an immune-privileged target organ, the eyes have important superficial and internal barriers, protecting them from physical and chemical damage from exogenous and/or endogenous origins that would cause injury to visual acuity or even vision loss. These anatomic, physiological and histologic barriers are thus a challenge for drug access and entry into the eye. Novel therapeutic concepts are highly desirable for eye treatment. The design of an efficient ocular drug delivery system still remains a challenge. Although nanotechnology may offer the ability to detect and treat eye diseases, successful treatment approaches are still in demand. The growing interest in nanopharmaceuticals offers the opportunity to improve ophthalmic treatments. Besides their size, which needs to be critically monitored, nanopharmaceuticals for ophthalmic applications have to be produced under sterilized conditions. In this work, we have revised the different sterilization Biology 2020,9, 336; doi:10.3390/biology9100336 www.mdpi.com/journal/biology Biology 2020,9, 336 2 of 18 and depyrogenation methods for ophthalmic nanopharmaceuticals with their merits and drawbacks. The paper also describes clinical sterilization of drugs and the outcomes of inappropriate practices, while recent applications of nanopharmaceuticals for ocular drug delivery are also addressed. Keywords: eye administration; ocular drug delivery; nanopharmaceuticals; ophthalmic treatments; sterilization; depyrogenation 1. Introduction The structure of the eye can be split into two main parts, namely, the anterior segment and the posterior segment [ 1 , 2 ]. As shown in Figure 1, the anterior segment includes the iris, ciliary body, cornea, conjunctiva, aqueous humor and lens, whereas the choroid sclera, neural retina, retinal pigment epithelium, vitreous humor and optic nerve belong to the posterior segment. Visual complications and blindness are usually related to several diseases of the posterior segment of the eye, in particular, in the retina. Biology 2020, 9, x 2 of 18 ophthalmic nanopharmaceuticals with their merits and drawbacks. The paper also describes clinical sterilization of drugs and the outcomes of inappropriate practices, while recent applications of nanopharmaceuticals for ocular drug delivery are also addressed. Keywords: eye administration; ocular drug delivery; nanopharmaceuticals; ophthalmic treatments; sterilization; depyrogenation 1. Introduction The structure of the eye can be split into two main parts, namely, the anterior segment and the posterior segment [1,2]. As shown in Figure 1, the anterior segment includes the iris, ciliary body, cornea, conjunctiva, aqueous humor and lens, whereas the choroid sclera, neural retina, retinal pigment epithelium, vitreous humor and optic nerve belong to the posterior segment. Visual complications and blindness are usually related to several diseases of the posterior segment of the eye, in particular, in the retina. Figure 1. Structure of the human eye and various form of drug delivery systems. Due to the complex anatomy and physiology of the eye, the delivery of therapeutic agents into its inner structures represents a great challenge. It is commonly accepted that an ideal delivery system should ensure effective drug levels over the timeframe of the treatment upon a single application [2]. The eye offers multiple routes through which ocular drugs can be delivered. Drug delivery to the anterior segment of the eye is currently achieved throughout topical and subconjunctival routes or injected intracamerally [2]. A topical eye drop is the most convenient and patient compliant route of drug administration, especially for the treatment of anterior segment diseases [3,4]. However, the ocular bioavailability is less than 5% of the topically applied dose [3,4]. The use of topical eye drops to achieve therapeutic drug concentration into the posterior segment thus represents a great challenge; several other pathways of drug administration, including periocular injections, intravitreal injections and systemic administration, are usually employed. Bearing in mind the fibrous composition and large surface area, the sclera offers less resistance to drug diffusion. Kang-Mieler's group has shown that molecules up to 70 kDa can readily penetrate the sclera, whereas only those below 1 kDa penetrate through the cornea [2]. As they have limited adhesion onto the ocular surface, topical eye drops often show insufficient drug retention; besides that, little is known about the effect of efflux transporters on the corneal surface. Several approaches to improve corneal resistance to nasolacrimal drainage have been Figure 1. Structure of the human eye and various form of drug delivery systems. Due to the complex anatomy and physiology of the eye, the delivery of therapeutic agents into its inner structures represents a great challenge. It is commonly accepted that an ideal delivery system should ensure effective drug levels over the timeframe of the treatment upon a single application [ 2 ]. The eye offers multiple routes through which ocular drugs can be delivered. Drug delivery to the anterior segment of the eye is currently achieved throughout topical and subconjunctival routes or injected intracamerally [ 2 ]. A topical eye drop is the most convenient and patient compliant route of drug administration, especially for the treatment of anterior segment diseases [ 3 , 4 ]. However, the ocular bioavailability is less than 5% of the topically applied dose [3,4]. The use of topical eye drops to achieve therapeutic drug concentration into the posterior segment thus represents a great challenge; several other pathways of drug administration, including periocular injections, intravitreal injections and systemic administration, are usually employed. Bearing in mind the fibrous composition and large surface area, the sclera offers less resistance to drug diffusion. Kang-Mieler’s group has shown that molecules up to 70 kDa can readily penetrate the sclera, whereas only those below 1 kDa penetrate through the cornea [2]. Biology 2020,9, 336 3 of 18 As they have limited adhesion onto the ocular surface, topical eye drops often show insufficient drug retention; besides that, little is known about the effect of efflux transporters on the corneal surface. Several approaches to improve corneal resistance to nasolacrimal drainage have been explored, such as the use of viscosity builders and corneal penetration enhancers by inhibiting p-glycoprotein. If, on the one hand, the effect of viscosity in liquid preparations is limited due to the clearance rate, on the other hand, the overly sensitive nature of the corneal and conjunctival tissues requires imposing caution to prevent toxicity [5,6]. Different conventional and innovative drug delivery systems, such as ointments, emulsions, aqueous gels, suspensions, nanoparticles, nanomicelles, dendrimers, liposomes, implants, contact lenses, nanosuspensions, microneedles and in situ thermosensitive gels, have been developed with the aim to overcome the ocular barriers and improve drug bioavailability in eye tissues [ 7 ]. Advances in nanotechnology have resulted in the development of ocular formulations based on biodegradable microparticles and nanoparticles, hydrogels and also implants, resulting the improvement of the bioavailability of several ocular drugs [2]. The knowledge of a patient’s hygienic habits is particularly important for clinical ophthalmologists, since pathogens have been identified even in the tears of asymptomatic patients [ 8 ]. Because of the intimate contact with the eye tissues, not only the drug delivery systems but also the ophthalmic tools used in clinical practice should be free from microorganisms. Sterilization is thus mandatory for both drug delivery systems and ophthalmic tools. For instance, the use of the ocular tonometer (i.e., for the measurement of the intraocular pressure by eye care professionals) is associated with the most common ophthalmic nosocomial outbreaks [ 9 , 10 ]. The test involves the touching of an anesthetized cornea by a tonometer tip that aims to record the amount of force required to flatten the cornea. To implement sanitization of this instrument, major guidelines are required, differing on procedures to disinfect tonometers. The recommendations from the World Health Organization indicate the use of 3–6% hydrogen peroxide for cleaning tonometer tips. However, according to the Guidelines for Disinfection and Sterilization in Healthcare Facilities, 3% hydrogen peroxide and 70% isopropyl alcohol are ineffective against adenoviruses. The adoption of disposable tonometer tips, particularly for patients suspected of having prion diseases, is recommended by the scientific literature, since it has been proven that no tonometer disinfectant is fully efficient against prions [9]. Nanopharmaceuticals for eye administration, known as complex systems, which are made of multiple components, should necessarily be sterile [ 11 – 13 ]. Thus, the manufacturing process must be sterile or terminal sterilization should be included in the process, while the further testing of nanopharmaceuticals in clinical trials is related to the approval of the production methods and quality assurance of the final product [ 11 , 12 , 14 ]. Besides the physical methods, such as irradiation, filtration or autoclaving, there are also chemical treatments, including the use of hydrogen peroxide, gas plasma, ethylene oxide and chemical vapor [ 15 , 16 ]. All of these methods, however, may have a negative influence on the physical/chemical characteristics of nanopharmaceutical dosage forms. The choice of the sterilization technique is instrumental to ensure antimicrobial safety and the extended shelf life of the product. Furthermore, well-optimized sterilization parameters highly protect the product against degradation and can limit the final amount of toxic residues released. Preformulation studies designed for the development of sterilized nanopharmaceuticals also need toensurethattheprocess canbescaledup[ 16 , 17 ]. Anotherchallengeinproducingnanopharmaceuticals is the fact that, apart from the sterilization, the product must be pyrogen free [ 17 ]. This is monitored by performing endotoxin assays on the drug products. Endotoxins trigger the immune system and activate the release of proinflammatory mediators which lead to endotoxin shock, tissue injury and sometimes death [ 18 ], outcomes that depend on the amount of endotoxin present [ 17 ]. Most nanopharmaceuticals interfere with the available endotoxin assays, which reduces the reliability of the test results. Therefore, other assays may be required. Non-sterilized conditions for nanopharmaceuticals can be beneficial, although it is important to start the production with sterile, pyrogen-free raw materials to ensure that production is carried out under microorganism-free conditions [ 16 ]. Since nanopharmaceuticals have Biology 2020,9, 336 4 of 18 revolutionized the way of producing new drug formulations and their administration, it is possible to overcome the ocular drug delivery barriers and improve ocular bioavailability of several drugs. In this review, we discuss the sterilization and depyrogenation methods, commonly used for ophthalmic nanopharmaceuticals, and their clinical applications. 2. Sterilization Methods of Ophthalmic Nanopharmaceuticals In order to prevent disease transmission related to the use of the product, the sterilization that refers to the destruction of microorganisms (e.g., viruses, fungi, spores) is recommended. The process using ethylene oxide (ETO) at a low temperature has been the most commonly applied since the 1950s for sterilizing temperature/moisture-sensitive ophthalmic healthcare facilities in the United States. Moreover, this method has been validated by the sterilizer manufacturer for the specific instruments, in relation to potential ocular toxicity, efficacy of sterilization and instrument functionality [10]. Different sterilization methods of ophthalmic nanopharmaceuticals are illustrated in Figure 2and they are further described in the sections below. Advantages and drawbacks of each method, as well as their most significant effects, are summarized in Table 1. Biology 2020, 9, x 4 of 18 2. Sterilization Methods of Ophthalmic Nanopharmaceuticals In order to prevent disease transmission related to the use of the product, the sterilization that refers to the destruction of microorganisms (e.g., viruses, fungi, spores) is recommended. The process using ethylene oxide (ETO) at a low temperature has been the most commonly applied since the 1950s for sterilizing temperature/moisture-sensitive ophthalmic healthcare facilities in the United States. Moreover, this method has been validated by the sterilizer manufacturer for the specific instruments, in relation to potential ocular toxicity, efficacy of sterilization and instrument functionality [10]. Different sterilization methods of ophthalmic nanopharmaceuticals are illustrated in Figure 2 and they are further described in the sections below. Advantages and drawbacks of each method, as well as their most significant effects, are summarized in Table 1. Figure 2. Different sterilization methods of ophthalmic nanopharmaceuticals. 2.1. Moist Heat Sterilization Using Autoclave An autoclave, based on moist heat sterilization, is a piece of equipment used for killing microorganisms [19]. Autoclaving, as an efficient method to inactivate bacteria, viruses and other biological material, is recommended for disposal regulated medical waste [20,21]. Depending on the high temperature (around 120 °C), chemical and structural changes in nanopharmaceuticals may occur. These modifications are mainly observed for nanopharmaceuticals that contain heat-sensitive drugs. Physical changes of nanopharmaceuticals, such size and morphology, can also be caused by their autoclaving, which can lead to variation of the amount of drug loaded in nanoparticles and the rate of drug release [16]. Both lipid-based and self-assembled nanoparticles, as well as those with a glass transition and melting point below 120 °C, are reported to be the most impacted by moist heat [16]. Both the increase in temperature during this process and the cooling will lead to rearrangement of nanoparticles’ molecular structure. Moreover, even in the presence of surfactants or stabilizers that reduce the risk of particles’ aggregation, the internal structure of the particle should not be affected by the nanopharmaceuticals’ sterilization method [16]. High temperatures used in autoclaving have a destructive influence on the nanoparticles based on matrix materials of low melting point. By reducing the temperature, the autoclaving time can be prolonged in order to counteract the degradation of the nanoparticle’s matrix induced by heat, ensuring the sterilization efficacy [16]. Vetten et al. (2014) [17] have summarized the challenges encountered in the sterilization of nanoparticles, for instance, the risk of aggregation, size increase of the nanoparticles and modifications on their surface charge. The authors demonstrated that all identified changes were dependent on the polymer or surfactant used in the production of particles, as well as on the Figure 2. Different sterilization methods of ophthalmic nanopharmaceuticals. 2.1. Moist Heat Sterilization Using Autoclave An autoclave, based on moist heat sterilization, is a piece of equipment used for killing microorganisms [ 19 ]. Autoclaving, as an efficient method to inactivate bacteria, viruses and other biological material, is recommended for disposal regulated medical waste [ 20 , 21 ]. Depending on the high temperature (around 120 ◦ C), chemical and structural changes in nanopharmaceuticals may occur. These modifications are mainly observed for nanopharmaceuticals that contain heat-sensitive drugs. Physical changes of nanopharmaceuticals, such size and morphology, can also be caused by their autoclaving, which can lead to variation of the amount of drug loaded in nanoparticles and the rate of drug release [ 16 ]. Both lipid-based and self-assembled nanoparticles, as well as those with a glass transition and melting point below 120 ◦ C, are reported to be the most impacted by moist heat [ 16 ]. Both the increase in temperature during this process and the cooling will lead to rearrangement of nanoparticles’ molecular structure. Moreover, even in the presence of surfactants or stabilizers that reduce the risk of particles’ aggregation, the internal structure of the particle should not be affected by the nanopharmaceuticals’ sterilization method [ 16 ]. High temperatures used in autoclaving have a Biology 2020,9, 336 5 of 18 destructive influence on the nanoparticles based on matrix materials of low melting point. By reducing the temperature, the autoclaving time can be prolonged in order to counteract the degradation of the nanoparticle’s matrix induced by heat, ensuring the sterilization efficacy [16]. Vetten et al. (2014) [ 17 ] have summarized the challenges encountered in the sterilization of nanoparticles, for instance, the risk of aggregation, size increase of the nanoparticles and modifications on their surface charge. The authors demonstrated that all identified changes were dependent on the polymer or surfactant used in the production of particles, as well as on the production method. Besides the physicochemical changes, the moist heat sterilization in autoclaving can also lead to changes in biological effects [22–24]. If nanopharmaceuticals are sterilized using the autoclave, it is necessary to verify if the autoclaved product suffered any thermal degradation. Autoclaving is considered acceptable in the case of no changes in the nanoparticles. Therefore, the morphology of nanoparticles, their long-term stability, particle size distribution, loading capacity and the rate of drug release should be evaluated in parallel [16]. Table 1. Overview of ophthalmic nanopharmaceutical sterilization methods. Sterilization Methods Effect Advantage Drawbacks References Autoclaving: high pressure steam bactericidal low cost chemical degradation, structural modification [16] Filtration: barrier physical retention drug thermally sensitive viscosity, size [17,25–27] Gamma irradiation: ionizing damage of genetic material viscous material, drug and adjuvant thermally sensitive, no residue, effective against bacteria, yeast, fungus chemical degradation, free radical, rate of drug delivery, gas formation, high cost [16,28] Gaseous ethylene oxide bactericidal low cost, drug and adjuvant thermally sensitive toxic residue, cascade of oxidation, chemical change [29] High hydrostatic pressure affect the cellular structures or functions bar-resistant nanoparticles (polymeric carriers) modifies adsorption, physical and chemical stability [30] Formaldehyde bactericidal, but highly toxic low cost toxicity (truncates proteins) and carcinogens, affect the re-dispersion [17,31,32] Gas plasma: oxide reduction effect antimicrobial low temperature, non-toxic oxidative, aggregation [15,17,30] 2.2. Sterile Filtration Sterile filtration is one of the most commonly used methods for the sterilization of nanoparticles, especially those that are heat labile and within a small particle size range [ 33 ]. However, the viscosity of the solution limits the nanopharmaceuticals that can be sterilized by filtration. Due to their small size, nanoparticles are able to penetrate through 0.22 µ m filters that may lead to distortion of the particles and/or loss of the loaded drugs. A selective retention of the active ingredient on the fiber is another inconvenience that can occur [ 16 ]. In turn, physically removing microorganisms from thermally and chemically sensitive liquids is commonly applied using 0.22 µ m membrane filters, thereby helping to protect the structure of nanoparticles [ 17 ]. Notwithstanding, this method cannot be used if the particles are larger/similar to the pore size of the filters since clogging may occur, resulting in a decreased yield [ 25 , 26 ]. In order to overcome this problem, the size of the nanoparticles should be designed to be less than 0.15 µ m. Nagai et al. (2014) have described the biopharmaceutical advantages of ophthalmic nanoparticles ranged between 60 and 80 nm [ 27 ]. When this sterile filtration method Biology 2020,9, 336 6 of 18 is used, nanopharmaceuticals must be evaluated for changes in particle size and distribution, drug loading, composition of the formulation and viscosity changes [16]. 2.3. Gamma Irradiation Gamma irradiation is indicated for nanopharmaceuticals that are thermosensitive, and can be used in the sterilization of sealed packaged products. Gamma irradiation is a type of ionizing radiation of short wavelength that carries enough energy to free electrons from atoms or molecules. It causes damage to the DNA or RNA of bacteria, fungi, yeasts and viruses [34,35]. Gamma irradiation can induce the degradation of nanoparticles, and/or physicochemical modifications, such as crosslinking and chain scission in the matrix of nanoparticles. These changes can influence the rate of drug release from the nanoparticles. The main changes observed after gamma irradiation of polymers and lipids are related to the gas formation, double bond, busted radicals, color formation, crystallinity, thermal transition and change in the surface hydrophilicity—hydrophobicity [ 28 ]. One of the major problems is the production of reactive free radicals. In particular, if water occurs during the process, it can lead to a series of free radical reactions. These radicals may interact both with the matrix of nanoparticles and with the loaded drug, resulting in physicochemical changes of the structure of the drug and in its interaction with the nanoparticles’ matrix [16]. The irradiation induced by degradation is affected not only by the chemical structure of the polymer, but also by its physical form [ 36 ]. Moreover, it is necessary to consider not only the influence of the gamma radiation on the polymers composing the nanoparticles but also on their physicochemical stability. The drug may be sensitive to the gamma irradiation, even if the polymeric material is resistant. The formulation buffer can also affect the peroxide formation. For each type of nanopharmaceutical formulation, establishment of the gamma irradiation sensitivity is required [ 16 ]. Several approaches exist to reduce the effects of gamma irradiation, such as optimization of the irradiation dose, removal of water by freeze-drying, using gamma radiation-resistant excipients, including free radical scavengers in the formulation, removing the oxygen or applying a vacuum during irradiation [ 16 ]. Monitoring the level of free radicals is necessary, while the toxicity profile of the nanopharmaceuticals must be fully tested [ 16 ]. Besides the identified shortcomings, gamma irradiation is still a method of interest for the sterilization of nanopharmaceuticals, because the results (e.g., shelf-life of the product, toxicity profile and induced responses) are not significantly changed in most cases [16,34]. 2.4. Other Irradiation Methods Several other irradiation methods, such as electron beam, X-ray radiation and UV light have also been proposed for the sterilization of nanoparticles. They are independent from heat and chemicals and do not leave residue after sterilization [ 17 ]. For electron beam sterilization, very high electron energy is used for a shorter duration when compared to gamma irradiation [ 17 ]. It is very effective, although it can damage the material being sterilized (similar to gamma irradiation). Some of the disadvantages of this method include changes in size, morphology, molecular mass and the pH of nanoparticles, degradation of antimicrobial and anti-sedimentation agents and changes in release profile, however, these changes do not always adversely affect the loaded drug [ 31 ]. Moreover, UV light irradiation is indicated for sterilization of surfaces using a germicidal lamp [ 31 ]. This method is effective in preventing bacterial growth, but it can only be applied if the nanoparticles’ properties remain unchanged [17]. 2.5. Gaseous Ethylene Oxide Gaseous ethylene oxide is usually used for devices that are not heat tolerant. This method works by alkalizing proteins and nucleic acids. Among some disadvantages, chemical changes in the drug or in the nanoparticles’ matrix, as well as the interactions between them, may be mentioned. Harmful gas residues may remain on the surface or within the nanoparticles, causing hemolysis. Additionally, Biology 2020,9, 336 7 of 18 in living organisms, ethylene oxide produces covalent adducts with DNA that cause mutagenic and genotoxic effects [ 37 ]. Some treated materials can generate toxic residues or set offa cascade of free radical reactions in the nanoparticles’ matrix material, in the drug or both. Therefore, after being sterilized, nanopharmaceuticals must be tested for residues of the gas and other toxic degradants [ 38 ]. The ethylene oxide can also promote changes in the rate of drug release, long-term stability and in the toxicological profiles of nanopharmaceuticals [ 16 ]. Friess and Schlapp reported aggregation and changes in the release profile of drug-loaded nanoparticles when sterilized with ethylene oxide, thereby demonstrating that this method can destructively influence nanopharmaceuticals [39]. 2.6. High Hydrostatic Pressure Sterilization It has been proven that a high hydrostatic pressure sterilization was ineffective in the process of spore elimination in high-pressure-resistant nanoparticles [ 16 ]. A high hydrostatic pressure up to 500 MPA for 30 min induces neither physical nor chemical damages, except on the surface modifiers that can be absorbed onto the nanoparticles. Brigger et al. (2003) have proposed high hydrostatic pressure as an innovative methodology for the sterilization of drug polymeric carriers, also underlying how further exploitation in this field is needed to develop novel protocols for spore inactivation [30]. 2.7. Formaldehyde Formaldehyde is an organic solution commonly used as a disinfectant and as a fixative [ 17 ]. It can be useful for materials that are sensitive to high temperatures, however, it is toxic and carcinogenic [ 28 ]. Sommerfeld et al. (1998) reported that aseptic manufacturing would be preferential over formaldehyde for sterilization purposes [32]. The chemical sterilization of poly(butylcyanoacrylate) nanoparticles by formaldehyde was shown to be inappropriate because of the difficulty in re-dispersing the freeze-dried particles obtained with different stabilizers, after formaldehyde treatment at 60 ◦C [32]. 2.8. Gas Plasma Gas plasma can be defined as an ionized gas that has properties of both gases and liquids. It has been shown to have a broad spectrum of antimicrobial effects [ 17 ], can be used at low temperatures and it is non-toxic [ 40 ]. Although its mechanism of action is not fully understood, it is believed that gas plasma is related to oxidation and reduction effects on microbial structures [ 41 ]. Some adverse effects, e.g., aggregation and alteration of the coating, have been observed due to the high oxidative nature of gas plasma [15]. 3. Endotoxin Contamination Bacterial endotoxin or lipopolysaccharide (LPS) is produced by Gram-negative bacteria and is known to be a potent inflammatory mediator causing septic shock syndrome, it can cause diffuse lamellar keratitis and is the main factor for toxic anterior segment syndrome (TASS). Gram-negative bacteria (predominantly Sphingomonas,Ralstonia and Pseudomonas) can form biofilms on the surface of objects and in formulations. Although Gram-negative bacteria can be destroyed by short-cycle sterilization methods, endotoxins are released from their bacterial cell walls and can remain on the surface of objects (e.g., ophthalmic instruments) and in the formulations. Endotoxins are heat-resistant and can remain biologically active, posing serious concerns for the safe use of such tools, including nanopharmaceuticals. The traditional test used to quantify endotoxin contamination is limulus amebocyte lysate (LAL). It has been used as an official test to replace the rabbit pyrogen test. The LAL test has three formats: chromogenic, turbidity and gel clot [ 17 ]. Although the formal acceptance requirements for the validity of LAL are in conformity, it is possible to find different results for the same nanoparticles. As concluded by Dobrovolskaia et al. [ 42 ], when these differences in LAL results are higher than 35%, the outcomes are verified in vivo using the rabbit pyrogen test. This problem can occur due to nanoparticle interference with the reactivity of the endotoxin in the LAL reaction [ 17 ]. Biology 2020,9, 336 8 of 18 Moreover, as marked by Smulders et al. [ 43 ], it is also important to include controls to help recognize the cause of negative results. The endotoxins cannot be removed by the traditional filtration method as they will easily pass through the membrane filter. Thus, other types of filtration are used in the pharmaceutical industry, such as chromatography and ultrafiltration, to remove them [ 18 ]. However, before using these methods, it is required to ensure that nanopharmaceuticals have no contaminants and do not interact with the columns [ 17 ]. Due to the complexity of nanoparticles and endotoxin, there is no efficient technique for their removal. The Food and Drug Administration (FDA) has recommended either applying high temperatures or high concentrations of acids/bases, but these conditions will likely affect the properties of nanopharmaceuticals. In this case, a contaminant-free production process is recommended [17]. Depyrogenation refers to the removal or inactivation of pyrogens. The depyrogenation method is chosen according to the procedure to be performed to remove endotoxin from nanopharmaceuticals [17,18,44–47] . Table 2shows methods used for depyrogenation and its main features. Table 2. Methods used to remove endotoxin (depyrogenation). Methods Principle of the Method Ultrafiltration It eliminates the endotoxin by molecular weight using ultra-fines (10,000 Daltons or greater). It shows relatively good endotoxin clearance. Reverse osmosis This process uses a filter and highly pressurized conditions. It captures 99.5% of endotoxin and ions or salts but allows water molecules through. It is commonly used to produce highly purified water. Two-phase partitioning In this system, an aqueous surfactant solution spontaneously separates into two predominantly aqueous, but immiscible, forms in an effective separation. Affinity chromatography This method acts to bind endotoxin through biding affinity using ligands such as DEAE Sepharose, poly-L-lysine and polymyxin-B. Moreover, this method can be affected by the pH range, temperature, flow rate and the number of electrolytes in the solution. Distillation Endotoxin is removed by the rapid evaporation of the water molecules and the persistence of the larger lipopolysaccharide molecules in the original environment. Adsorption The endotoxin molecule is attracted to the activated carbon bed. This mechanism is less efficient and is affected by several environmental factors. Acid-base hydrolysis Occurs in the binding of lipid A with the polysaccharide nucleus. The isolated molecule is insoluble in an aqueous medium. The main acids used are HCl and glacial acetic acid diluted. Plasma discharge The UV radiation used in this process is responsible for the inactivation of spores. The main advantage is the possibility of the operation of the process at moderate temperatures, allowing the treatment of heat-degradable materials. Oxidation Depyrogenation occurs by peroxidation of the fatty acid in the lipid A region (e.g., using hydrogen peroxide) Ethylene oxide The process is performed in a heated, pressurized chamber, but at a lower temperature. The depyrogenation process occurs by nucleophilic substitution in the glucosamine of lipid A. Moist heat Traditional autoclaving does not destroy endotoxins. However, when combined with hydrogen peroxide and pressure, it is effective. Dry heat Endotoxins are destroyed by exposure to high temperature. 4. Sterilization of Ophthalmic Nanopharmaceuticals Several sterilizations methods of ophthalmic pharmaceuticals based on different types of nanoparticles (lipid-based, silver, magnetic or gold nanoparticles) are here described and summarized in Figure 3. Biology 2020,9, 336 9 of 18 Biology 2020, 9, x 9 of 18 Figure 3. Different types of nanoparticles used as ophthalmic nanopharmaceuticals. 4.1. Solid Lipid Nanoparticles Solid lipid nanoparticles (SLNs) have been proposed as colloidal drug carriers for several administration routes [48–51], including eye administration [3,4,52–56]. They offer several advantages, such as bioacceptable and biodegradable composition, small size, no toxicity, high loading capacity or controlled drug delivery [54–58]. SLNs can be produced using several methods, among which high-pressure homogenization and the microemulsion method at high temperatures may act as sterilizing approaches [59,60]. Cavalli et al. (1997) [61] have developed SLNs by dispersing warm oil-in-water microemulsions in a cold aqueous medium under mechanical stirring [61]. Upon lipid recrystallization, a dispersion of SLNs is obtained from the oil droplets. To obtain dry products and provide longer stability, SLN dispersions should be lyophilized, although this process can also lead to changes in the mean size and shape of nanoparticles [61]. Several sterilization approaches have been used in lipid nanoparticles. The autoclaving of SLNs for 15 min at 121 °C under pressure of 2 bar has been carried out by Cavalli et al. according to the European Pharmacopoeia II, showing that SLNs were stable during the process and maintained a spherical shape and narrow size distribution [61]. Moreover, no particles larger than 1 µm were recorded in the samples stored at 4 °C for more than a year. The high temperatures reached during autoclaving presumably created a hot oil/water microemulsion, which was then recrystallized in the form of SLNs within the nanometer size range [61]. No differences were observed between a drugloaded SLN compared with the drug-free SLN [61]. The average diameter (Z-Ave) and polydispersity index (PI) slightly increased, while the zeta potential (ZP) mostly remained the same [61]. To ensure a spherical shape without any significant increase in the size or particle size distribution, SLNs can be sterilized by autoclaving. Although the stability of the lipid matrix should not be affected, SLNs loaded with heat-resistant drugs should be sterilized by a transition temperature above that of the autoclaving [16]. 4.2. Hydrogels Containing Silver Nanoparticles Hydrogels (HGs) are described as three-dimensional and crosslinked polymer networks. They are able to retain large quantities of water [14]. Among their most important features, their high biocompatibility, possibility of modulating mechanical properties and production facilities, are highlighted [62]. Figure 3. Different types of nanoparticles used as ophthalmic nanopharmaceuticals. 4.1. Solid Lipid Nanoparticles Solid lipid nanoparticles (SLNs) have been proposed as colloidal drug carriers for several administration routes [ 48 – 51 ], including eye administration [ 3 , 4 , 52 – 56 ]. They offer several advantages, such as bioacceptable and biodegradable composition, small size, no toxicity, high loading capacity or controlled drug delivery [ 54 – 58 ]. SLNs can be produced using several methods, among which high-pressure homogenization and the microemulsion method at high temperatures may act as sterilizing approaches [ 59 , 60 ]. Cavalli et al. (1997) [ 61 ] have developed SLNs by dispersing warm oil-in-water microemulsions in a cold aqueous medium under mechanical stirring [ 61 ]. Upon lipid recrystallization, a dispersion of SLNs is obtained from the oil droplets. To obtain dry products and provide longer stability, SLN dispersions should be lyophilized, although this process can also lead to changes in the mean size and shape of nanoparticles [61]. Several sterilization approaches have been used in lipid nanoparticles. The autoclaving of SLNs for 15 min at 121 ◦ C under pressure of 2 bar has been carried out by Cavalli et al. according to the European Pharmacopoeia II, showing that SLNs were stable during the process and maintained a spherical shape and narrow size distribution [ 61 ]. Moreover, no particles larger than 1 µ m were recorded in the samples stored at 4 ◦ C for more than a year. The high temperatures reached during autoclaving presumably created a hot oil/water microemulsion, which was then recrystallized in the form of SLNs within the nanometer size range [ 61 ]. No differences were observed between a drug-loaded SLN compared with the drug-free SLN [ 61 ]. The average diameter (Z-Ave) and polydispersity index (PI) slightly increased, while the zeta potential (ZP) mostly remained the same [61]. To ensure a spherical shape without any significant increase in the size or particle size distribution, SLNs can be sterilized by autoclaving. 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