Current Situation and Challenges in Vitreous Substitutes

REVIEW www.mbs-journal.de Current Situation and Challenges in Vitreous Substitutes Cristina Mondelo-García, Enrique Bandín-Vilar, Laura García-Quintanilla, Ana Castro-Balado, Eva M. del Amo, María Gil-Martínez, María José Blanco-Teijeiro, Miguel González-Barcia, Irene Zarra-Ferro, Anxo Fernández-Ferreiro,* and Francisco J. Otero-Espinar* Vitreo-retinal disorders constitute a significant portion of treatable ocular diseases. These pathologies often require vitreo-retinal surgery and, as a consequence, the use of vitreous substitutes. Nowadays, the vitreous substitutes that are used in clinical practice are mainly divided into gases (air, SF6,C 2F6,C 3F8) and liquids (perfluorocarbon liquids, silicone oils, and heavy silicone oils). There are specific advantages and drawbacks to each of these, which determine their clinical indications. However, developing the ideal biomaterial for vitreous substitution continues to be one of the most important challenges in ophthalmology, and a multidisciplinary approach is required. In this sense, recent research has focused on the development of biocompatible, biodegradable, and injectable hydrogels (natural, synthetic, and smart), which also act as medium and long-term internal tamponade agents. This comprehensive review aims to cover the main characteristics and indications for use of the extensive range of vitreous substitutes that are currently used in clinical practice, before going on to describe the hydrogels that have been developed recently and which have emerged as promising biomaterials for vitreous substitution. C. Mondelo-García, E. Bandín-Vilar, L. García-Quintanilla, A. Castro-Balado, M. González-Barcia, I. Zarra-Ferro, A. Fernández-Ferreiro Pharmacy Department University Clinical Hospital of Santiago de Compostela (SERGAS) Santiago de Compostela 15706, Spain E-mail: [email protected] C. Mondelo-García, E. Bandín-Vilar, L. García-Quintanilla, A. Castro-Balado, M. González-Barcia, I. Zarra-Ferro, A. Fernández-Ferreiro Pharmacology Group Health Research Institute of Santiago de Compostela (FIDIS) Santiago de Compostela 15706, Spain The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/mabi.202100066 © 2021 The Authors. Macromolecular Bioscience published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. DOI: 10.1002/mabi.202100066 1. Introduction The vitreous humour is a transparent gel present between the lens and the retina. It has a volume of around 4 mL and occupies 80% of the eye volume.[1,2] It weighs around 4 g and contains approximately 99% water, only adhering to ocular structures in the following parts: the macula, the optic nerve disc, and the anterior border of the area surrounding the retina.[2] Vitreo-retinal disorders constitute a significant portion of treatable ocular diseases. The vitreous humour often becomes dysfunctional due to opacification, liquefaction or physical collapse, as a result of inflammatory diseases, developmental abnormalities, vitreous hemorrhage, tumors, diabetes, or degenerative processes. In addition, vitreous damage can also be caused by intraocular foreign bodies or trauma.[3] The vitreous humour determines the clarity of vision meaning E. M. del Amo School of Pharmacy, Faculty of Health Sciences University of Eastern Finland Kuopio 70211, Finland M. Gil-Martínez, M. J. Blanco-Teijeiro Ophthalmology Department University Clinical Hospital of Santiago de Compostela (SERGAS) Santiago de Compostela 15706, Spain F. J. Otero-Espinar Department of Pharmacology, Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy University of Santiago de Compostela (USC) Santiago de Compostela 15782, Spain E-mail: [email protected] Macromol. Biosci. 2021, 2100066 2100066 (1 of 15) © 2021 The Authors. Macromolecular Bioscience published by Wiley-VCH GmbH www.advancedsciencenews.com www.mbs-journal.de therefore that, if not treated properly, these disorders can cause blindness. Vitreous substitutes are crucial adjuncts during vitreoretinal (VR) surgery for retinal diseases.[2,4,5] Nowadays, the most common agents that are used as vitreous substitutes in clinical practice boast certain advantages, including chemical inertness and optical clarity, nonetheless, there are also many limitations to their use.[2] In this sense, the development and characterization of new vitreous substitutes have played an important role in vitreo-retinal surgery. Despite the fact that considerable efforts have been made in developing new biomaterials for vitreous substitution in search of the perfect alternative that is able to overcome the disadvantages presented by the currently available substances, further research must be performed. In this regard, developing the ideal biomaterial for vitreous substitution continues to be one of the most significant challenges in ophthalmology. Given the complexity of this matter, a multidisciplinary approach, which involves ophthalmic surgeons, pharmacists, chemists, and experts in biomaterials will be necessary if we want to overcome this problem. Our aim is to discuss the main characteristics and applications of the wide variety of vitreous substitutes that are currently used in clinical practice, before going on to address the development of new hydrogels that have been presented as promising alternatives for the optimization of vitreous substitution. 2. Composition and Functions of the Vitreous The vitreous is a gelatinous structure that is composed of 98% water.[3,4] It protects the adjacent structures and tissues from traumas, as well as permitting the circulation of nutrients and solutes, and controlling the oxygen tension within the eye. It helps to maintain the shape of the ocular globe, as well as keeping the crystalline lens and the retina in their place.[3,6] The main components of the vitreous humour, as well as the major characteristics of the aforementioned components are outlined below. 2.1. Proteins Proteins represent a small percentage of the overall content of the vitreous components. The majority of the soluble proteins are albumin (40%), with the other important components including immunoglobulins and iron-binding proteins such as transferrin, which helps to reduce iron toxicity in the case of small hemorrhages.[1,6,7] With regards to insoluble proteins, collagens are the most abundant. There are different types of proteins which play a major role in the vitreous structure.[4,8] 2.2. Glycosaminoglycans (GAGs) GAGs, which are extracellular polysaccharides are a key component of the vitreous structure, and these are mainly divided into three types—hyaluronic acid, heparan sulfate, and chondroitin sulfate.[1] 2.2.1. Hyaluronic Acid (HA) HA is a major component of the vitreous, forming 3D structures with collagen. The fact that it does not contain sulfate makes it distinguishable from the other GAGs, and this also means that it does not attach to proteins to form a proteoglycan.[1] The highest concentrations of hyaluronan molecules are found in the posterior vitreous cortex.[6,9–11] The HA preparations consist of molecules with greater variability in terms of hydrodynamic size, and these are a relevant component in determining vitreous viscosity.[12] 2.2.2. Chondroitin Sulfate (CS) CS is a sulfated GAG, which consists of a chain of alternating sugars (N-acetylgalactosamine and glucuronic acid). It constitutes a major component of the extracellular matrix and is also present in the vitreous, appearing in the form of the proteoglycans versican and type IX collagen.[4,11] CS is used to preserve the integrity of the vitreous and provide resistance against compression.[1] 2.2.3. Heparan Sulfate (HS) HS is a renewable proteoglycan, which ensures that there is adequate spacing between the collagen fibrils, nonetheless, small amounts of HS are present in the vitreous.[13] It also enhances the regulation of angiogenesis and blood coagulation, as well as maintaining vitreo-retinal adhesion.[4] 2.3. Glucose, Lactic Acid, and Antioxidants Due to the important role that the vitreous plays in the cellular metabolism of ocular tissues, its components include several substances that act as subtracts for metabolism, such as glucose and lactic acid, which are necessary to support the metabolism in the surrounding tissues.[4,6] In addition, the vitreous acts as a reservoir of glucose for the ciliary body.[14] On the other hand, ascorbic acid is a crucial antioxidant for lens and retinal metabolism, in particular it is used as a metabolic buffer in potassium homeostasis. Furthermore, it may also inhibit neovascularization and increase the proliferation of hyalocytes.[15–17] 2.4. Cells and Enzymes There are three types of cells that are found in the vitreous body: hyalocytes, fibrocytes/fibroblasts, and macrophages.[6,18,19] The main functions of these cells are related to the creation, regulation, and degradation of the vitreous matrix. Several enzymes have been isolated, which include hyaluronidase, serine proteases, and renin-angiotensin-converting enzyme.[20–22] 3. Vitreous Substitutes in Clinical Practice Vitreous substitutes must have physical and biological properties that make them suitable for use in clinical practice. In terms of their physical properties, ideal vitreous substitutes will be hydrophilic and insoluble in water, easy to manipulate during surgery, clear and transparent to facilitate visualization, and Macromol. Biosci. 2021, 2100066 2100066 (2 of 15) © 2021 The Authors. Macromolecular Bioscience published by Wiley-VCH GmbH www.advancedsciencenews.com www.mbs-journal.de Figure 1. Gas injection in pneummoretinopexy. A) Example of a superior detachment and injection with the patient lying in the supine position (face-up). B) The patient is placed in an upright position and the bubble tamponades the break. Created with BioRender.com. they will also have a refractive index that is similar to the human vitreous. In addition, these must remain stable when injected through a small syringe, as well as ensuring adequate surface tension in the attempt to seal the retinal break.[2,23] On the other hand, and in terms of their biological properties, the ideal vitreous substitutes will be biodegradable and biocompatible, biologically and chemically inert, and they will not block the aqueous drainage. Furthermore, they must be nontoxic to retinal tissues.[6,23] An ideal vitreous substitute does not yet exist; all of them offer advantages and drawbacks, which may or may not make them suitable depending on the clinical situation. Those currently used in clinical practice are divided into different categories based on different properties. Specifically, these are divided into gases (air, SF6,C 3F8,andC 2F6) and liquids (salt solution, silicon oils (SO), perfluorocarbon liquids, and semi fluorinated alkanes). The characteristics of each individual group, as well as their clinical indications have been outlined in detail in the following sections. 3.1. Gases Ohm was the first to describe intraocular gas use in 1911.[24] Several indications for its use were described, with the first of these being its use as an internal tamponade.[25] It also proves useful in unfolding and folding the retina, as well as in improving its postoperative visualization and replacing the globe volume to prevent fluid movement into retinal breaks. Gas is also used in several techniques: pneummoretinopexy (Figure 1); the pars plana vitrectomy for retinal detachment (RD), with and without scleral buckle, to flatten the retina; subretinal blood displacement in macular hemorrhages; and postvitrectomy liquid–gas exchange in previously vitrectomized eyes.[26] Postoperative care is very important in these patients. Prone (face-down) positioning must commence as soon as possible after the vitrectomy procedure has been performed. This is crucial in preventing any shift of the reattached retina. Moreover, it decreases the contact between the posterior surface of the lens with the gas bubble in phakic patients (the bubble is situated towards the retina); therefore, reducing the risk of cataract formation. Positioning may also vary depending on the location of the retinal break, usually lying on the opposite side of the break. The most widely used intraocular gases are sulfur hexafluoride (SF6) and perfluoropropane (C3F8). These are nontoxic and inert gases that are insoluble in the aqueous humour, and they boast a lower water solubility than nitrogen therefore allowing them to expand.[27] The decision as to which one of these gases is to be used will be based on the tamponade duration and the surgeon’s preferences, taking into consideration the type and location of the retinal break.[28] It is generally accepted that SF6can be used in uncomplicated primary RD. However, if a final tamponade action is necessary, based on the results from “The Silicone Study”, in which the results in patients with RD and proliferative vitreoretinopathy (PVR), the pars plana vitrectomy with either C3F8gas or SO tamponades were favorable.[29] However, the clear advantage of using gases is that it is not necessary to remove them. There are three stages in the absorption of the intraocular gas: expansion of the gas when injected, nitrogen equilibrium, and dissolution.[30] Gases may be absorbed by diffusion across the retina into the blood stream, or they may be dissolved into the aqueous humour, and removed through the anterior chamber.[31] The absorption of these gases demonstrates first-order pharmacokinetics, with small volumes lasting in the intraocular cavity for days or weeks depending on the gas used.[30] Knowledge of the kinetics of these gases, as detailed in Table 1, comes mostly from animal models and small studies in which indirect measurements have been taken in humans.[30–35] These studies may not be representative of current practice, especially with regards to the duration of the air bubble in the posterior chamber of the eye, which is perceived to be longer in clinical settings than in theoretical ones.[36,37] The half-life of intraocular gases is shorter in aphakic eyes than in phakic eyes, due to the increased convection in the vitreous cavity.[38] Following the vitrectomy procedure, convection currents appear in the aphakic eye, which accelerate the absorption rate. In phakic eyes with normal vitreous, there are much fewer convection currents; therefore, the expansion and absorption of long-acting gases is slower.[30] 3.1.1. Air Room air tamponade applied using non-expansible gas begins to shrink immediately after injection as it dissolves in the vitreous.[28] This offers an advantage over other long-lasting gases as it requires shorter prone positioning, allowing for a faster recovery of vision and less adverse effects.[41] The half-life of a room air tamponade was 1.6 d in phakic eyes[30] with a longevity of 5 d, but recent clinical impressions have suggested that air remains in the vitreous cavity for a longer period of time. In a recent retrospective cohort study, a half-life of 3.3 d with a longevity of 11.4 d was determined.[37] 3.1.2. Other Gases: SF6,C 2F6,C 3F8 Sulfur hexafluoride (SF6) and perfluoropropane (C3F8) are the most commonly used intraocular gases in clinical practice, compared to hexafluoroethane (C2F6) which is much less frequently used.[36] Macromol. Biosci. 2021, 2100066 2100066 (3 of 15) © 2021 The Authors. Macromolecular Bioscience published by Wiley-VCH GmbH www.advancedsciencenews.com www.mbs-journal.de Table 1. Pharmacokinetics of intraocular gases. Gases Nonexpansile concentration Duration of maximal expanded volume [h] Half-life [d] Duration of air bubble [d] Indications Rabbits Humans Rabbits Humans Animals Humans Air – Nonexpansible – 1.6a) 0.9b) 5–6 10.7– 11.4c) - Simple cases in which a short duration is required SF6(sulfuric hexafluoride) 20% 24–48 21 – 2.6a) 2.4b) 8–11 18a),c) - RD (Retinal detachment) with superior breaks - RD with inferior breaks - Flat RD in the case of meticulous vitreous dissection. - Following macular hole surgery C2F6(hexafluoroethane) 16% 72 27 2 – 16 34.5a),c) Not approved by FDA C3F8(perfluoropropane) 12% 72–96 30 6 5.7a) 4.5b) 4.3c) 28 67.7a),c) - RD and multiple breaks - RD with superior giant tear - RD with proliferative vitreoretinopathy (PVR) - Failed prior RD surgery - Persistent subretinal fluid - Following macular hole surgery - Pneumatic displacement of subretinal blood clot Ref. [28] [33,39] [36] [31] [30] [32,34] [36,37,40] [25] a) Referring to phakic eyes; b) Referring to aphakic eyes; c) Referring to pseudo-phakic eyes. Given that these gases boast a lower water solubility than nitrogen, they tend to expand to at least twice the volume of the gas injected, as a result, nonexpansile or minimally expansile mixture of gas is preferable in order to prevent adverse effects such as intraocular pressure elevation (IOP).[28] Water solubility varies depending on the carbon chain length. The longer the carbon chain, the lower the solubility in water; therefore, resulting in a longer intraocular longevity:[42] A mixture of 10% C3F8had a half-life of 5.7 d in phakic and 4.3 d in pseudophakic eyes.[30] A mixture of 20% SF6had a half-life of 2.8 d in phakic eyes. The half-life of C2F6was not measured; however, a longevity of 34 d was determined in the vitreous cavity.[36] Certain postoperative complications have been reported following the use of intraocular gas, however, most of these can be prevented by taking greater care when undertaking the surgical procedure. For example, gas could go under the retina although this is preventable, or gas could become entrapped at the injection site. One of the most frequent complications is the formation of cataracts due to the gas coming into contact with the crystalline lens.[43] Raised intraocular pressure may occur, but this tends to happen on the first postoperative day, and its cause has been attributed to the expansion of the bubble or to an overfilled eye. On the contrary, hypotony may occur if there is any gas leakage from the sclerotomies. Other complications include the presence of gas in the anterior chamber, secondary corneal decompensation,[44] which is more frequent in aphakic patients, and non-intact posterior capsule. In pseudophakic patients, intraocular lens capture may occur due to it being pushed forward into the anterior chamber.[45,46] 3.2. Liquids Different liquid vitreous substitutes are used in clinical practice. The main groups and their major characteristics are depicted in Table 2. 3.2.1. Salt Solutions Salt solutions have similar characteristics to aqueous humour in terms of their density, refractive index and transparency.[3] In the clinical setting, these are used on a temporary basis during the exchange with air or liquids as their low surface tension means that these do not have tamponade properties.[4] 3.2.2. Silicon Oil Silicon oil (SO) is a polymerized siloxane with organic side chains. It belongs to the class of synthetic organosilicon compounds and is a repetition of the –[R2Si–O]– group in which R is the organic side chain.[47] Specifically, SOs used as vitreous substitutes are polymers of polydimethylsiloxane (PDMS). In contrast to silicone rubber, polymer chains are shorter, and given the lack of chemical cross-linking between them, these present in a liquid form. They are hydrophobic substances with a specific gravity, which is slightly lower than water, and a refractive index that is higher than that of the vitreous.[48] These are available in different viscosities, which is measured in centistokes (cSt), and which ranges from 1000 to 5000 cSt in clinical practice. Macromol. Biosci. 2021, 2100066 2100066 (4 of 15) © 2021 The Authors. Macromolecular Bioscience published by Wiley-VCH GmbH www.advancedsciencenews.com www.mbs-journal.de Table 2. Physical properties of liquid vitreous substitutes and clinical indications. Product Specific gravity [g cm−3] Viscosity [cSt] Refractive index Indications A. SALT SOLUTIONS BSS 1 Temporary replacement during air/oil exchange B. SILICON OILS SO 1000 cSt 0.97 1000 1.4 Complex RD associated with PVR Giant tear, RD with PVR Traumatic RD with PVR Recurrent RD with breaks involving the lower quadrants RD associated with severe proliferative diabetic retinopathy RD associated with macular hole in pathologic myopia Pediatric RD RD associated with viral retinitis Posttraumatic endophthalmitis SO 2000 cSt 0.97 2000 1.4 SO 5000 cSt 0.97 5000 1.4 C. PERFLUOROCARBON LIQUIDS Perfluorooctane (C8F18) 1.76 0.69 1.27 Primary Rhegmatogenous RD Complicated RD with PVR Giant tear RD with PVR RD associated with disc coloboma, Dislocated lens, Suprachoroidal hemorrhage Perfluorodecalin (C10F18) 1.94 2.7 1.33 Perfluoroperhydrophenanthrene (C14F24) 2.03 8.03 1.31 Octafluoropropane (C3F8) 1.6 0.465 1.22 iv.SEMIFLUORINATED ALKANES Perfluorohexyloctane (F6H8) 1.35 3.44 1.387 D. SFA-SO combinations (a) Double fillings (b) Heavy Silicon Oils Densiron-68 1.06 1387 1.39 RD associated with inferior tears and PVR Oxane HD 1.02 3300 1.4 HWS 46–3000 1.12 2903 1.37 Since the 1960s, SO have been used as short and long-term vitreous substitutes because of their transparency, low surface tension, buoyancy, and low toxicity. The role of SO in clinical practice was defined by “The Silicone Study”,[49,50] a multicentre prospective randomized clinical trial that compared the effect of SO to long-acting intraocular gases (SF6and C3F8) in the management of complex RDs associated with severe PVR. Globally, SO was demonstrated to be more effective than SF6, and equally as effective as C3F8in reattaching the retina.[49] In addition, SO and C3F8produced very similar results in terms of the improvement of visual function and the low complication rates. Furthermore, the ophthalmologist‘s preference or the need for the patient to take a flight soon after the intervention could be reasons for using SO.[1] The use of SO in giant tears without PVR is still being debated. In this sense, good anatomic success has been reported with SO and gases. Generally speaking, SO is the most used agent in Europe, while in the United States some ophthalmologists still have a preference for intraocular gas.[51] SO tamponade tends to be administered at the primary vitrectomy for traction RD associated with severe proliferative diabetic retinopathy.[52] However, to date, no clinical trials have adequately evaluated its efficacy in this use. In addition, with regards to the treatment of RDs in viral retinitis, SO offers long-term internal tamponade, therefore decreasing the risk of re-detachment.[53,54] Regarding the pediatric population, the main indications for the use of SO tamponade are RDs associated with retinopathy of prematurity, trauma, congenital anomaly, and myopia. In the case of severe traumatisms, SO internal tamponade may help to flatten the retina and prevent hemorrhage, which would increase the risk of PVR.[55] Finally, it has been argued that SOs have certain antimicrobial activity, which is why they are usually used as a tamponade in posttraumatic endophthalmitis cases.[56] With regards to the disadvantages of SOs, these include the need for optical adjustments to be made due to the different refractive index when compared to the natural vitreous body, and the less effective nature of the use of the tamponade in treating inferior retina breaks due to its low specific gravity.[57,58] Furthermore, serious complications such as retinal toxicity,[59] optical neuropathy,[60] or glaucoma [61] have been reported with the use of SO, some of which are related to the emulsification of SO, especially in long-term use. This emulsification leads the original SO bubble to break down into smaller droplets, resulting in retinal inflammation by inducing a macrophagic response.[62] SOs must be removed as soon as they have fulfilled their purpose, and when it is established that further retention could increase the risk of complications (Figure 2). This removal is generally recommended within a six-month period after the intervention. Macromol. Biosci. 2021, 2100066 2100066 (5 of 15) © 2021 The Authors. Macromolecular Bioscience published by Wiley-VCH GmbH www.advancedsciencenews.com www.mbs-journal.de Figure 2. Silicone oil (SO) removal through active suction with machine assistance. Created with BioRender.com. 3.2.3. Perfluorocarbon Liquids Perfluorocarbon liquids (PFCLs) are fluorine-substituted hydrocarbons, which are clear, colorless and odorless liquids. These synthetic compounds are characterized by a high specific gravity, between 1.7 and 2.1 g cm−3, twice as high as water, and their refractive index is similar to that of the vitreous humour.[63] They are insoluble in water and poorly soluble in SOs. The most commonly used PFCLs in clinical practice are perfluorodecalin (C10F18), perfluoroperhydrophenanthrene (C14F24), perfluorooctane (C8F18 and octafluoropropane (C3F8).[4] Historically, these were first investigated as blood substitutes due to their extensive capacity for transporting and releasing oxygen and carbon dioxide.[64] In the clinical setting, the use of PFCLs has improved the visual outcomes and the anatomic success rate in PVR surgical procedures.[65–68] PFCLs provide the best currently available internal tamponade during membrane dissection. Similarly, the use of PFCLs in treating giant-tear RD has improved the anatomic success rate by over 90%.[66] The PFCLs allow for the repositioning of the folded flap, enabling direct PFCL-SO exchange in order to prevent the posterior flap from slipping. PFCLs also offer several advantages in the treatment of traumatic RD, including the stabilization of the retina during the vitrectomy procedure, the displacement of preretinal, subretinal, and suprachoroidal blood, the elimination of incarcerated vitreous or retina, and the maintenance of a transparent medium for visualization during surgery.[69] The use of PFCLs in other ophthalmic pathologies has already been proved, for example in cases of RD associated with diabetic retinopathy,[70] detachment associated with disc coloboma,[71] detachment from retinopathy of prematurity,[72] vitrectomy for endophthalmitis, displacement of submacular hemorrhage during surgical drainage, and the excision of subretinal membranes.[73] Figure 3. Perfluorocarbon liquid (PFCL) – silicone oil (SO) exchange. SO is filled progressively superiorly, while PFCL is extruded through the flute needle placed within the PFCL bubble. Created with BioRender.com. With regards to the safety of PFCLs, in recent years, several cases of retinotoxicity caused by perfluorooctane have been reported worldwide. In this regard, it is necessary for strict protocols to be established to determine the cytotoxicity of intraocular medical devices in order to ensure the adequate quality of these products.[74] Nowadays, their use is limited to the intraoperative setting because of their long-term toxicity, and as a result they have been exchanged with SO (Figure 3) or another long-term vitreous substitutes.[75–77] 3.2.4. Semifluorinated Alkanes Semifluorinated alkanes (SFAs) were identified in the 2000 s as an alternative to PFCLs given the presumption that the latter could cause retinal toxicity due to their high specific density. Moreover, SFAs maintain properties such as inertness, biocompatibility, interface tensions, etc., containing both perfluorocarbon and hydrocarbon segments. They have lower densities compared to PFCLs, ranging from 1.1 to 1.7 g cm−3, and they are soluble in PFCLs, hydrocarbons and SO. They also have very low surface and interface tensions.[76] The shorter the perfluoroalkyl chains and/or the longer the alkyl chain, the more toxic the semifluorinated alkanes are. Impurities containing –CHF groups must also be taken into account, given that hydrogen fluoride groups can be eliminated in the presence of nucleophilic bases, resulting in toxic alkenes.[76] SFAs were initially used as SO solvent, and later as temporary endotamponades when it was observed that SO was ineffective.[78] The most common problems related to the use of SFAs are cataracts and emulsification. Nowadays, SFAs tend to be mixed with SO. Macromol. Biosci. 2021, 2100066 2100066 (6 of 15) © 2021 The Authors. Macromolecular Bioscience published by Wiley-VCH GmbH www.advancedsciencenews.com www.mbs-journal.de 3.2.5. Silicon Oils and Semifluorinated Alkanes Combination The combined use of SO and SFAs tamponade agents has been widely studied, with the idea of bringing together the high viscosity of SO and the high specific gravity of SFAs. This mixture generates vitreous substitutes that boast good tamponade properties and minimal emulsification.[79] Depending on the proportion of SO and SFAs included in the mixture, it is possible to obtain homogeneous solutions (heavy silicone oils) or separated solutions (double fillings).[80] Heavy silicone oils—Heavy silicone oils (HSO) are homogeneous solutions that are heavier than water and that are formed by combining SO and SFAs. In clinical practice, these are used for treating complicated RDs, especially those which involve inferior PVR. There are three prefabricated HSOs currently on the market: Densiron 68, Oxane HD, and HWS 46–3000. Densiron 68 (Fluoron Co, Ulm, Germany) is a mixture of 30.5% SFA F6H8(perfluorohexyloctane) with 69.5% SO, 5000 cSt.[78] By adding SO, the viscosity of F6H8increases from 2.5 to almost 1400 mPa s, reducing its dispersion tendency, which is believed to cause the problems derived from the long-term use of F6H8. It has a specific gravity of 1.06 g cm−3and a refractive index of 1.387. Oxane HD (Bausch & Lomb, Toulouse, France) is a mixture of 88.1% Oxane 5700, a 5000 mPa s SO, with 11.9% RMN3, a partially fluorinated olefin. It has a slightly superior specific gravity and refractive index than water, 1.02 g cm−3and 1.4, respectively. It is the most viscous, 3800 mPa s, and least heavy HSO of the three products. Because of its lower specific gravity, slightly higher re-detachment rates were found when using this HSO.[81] The last commercialized HSO is called HWS 46–3000, which is a mixture of 45% ultrapurified SO 100 000 (viscosity 97 100 mPa s and specific gravity 0.977 g cm−3) and 55% perfluorobutylhexane (F4H6) a semifluorinated alkane (viscosity 1.28 mPa s and specific gravity of 1.254 g cm−3). The resulting solution (specific gravity of 1.105 g cm−3and a viscosity of 3109 mPa s) is homogeneous and stable in the presence of water, air or PFCLs. It is the heaviest and most viscous mixture of the three. In the pilot study by Rizzo et al., high success and low complication rates were achieved when HWS 46–3000 was used as a long-term tamponade (1–3 months), even though, due to its higher viscosity, handling this substance may be more difficult, e.g., when removing it.[75] Densiron 68, Oxane and HWS 46–3000 have shown promising results in the treatment of RDs associated with inferior tears.[82] However, in a prospective, multicentered, randomized controlled trial (HSO Study) that compared the effect of heavy tamponade (Densiron 68) and conventional SO in eyes with inferior and posterior PVR grade C or above, researchers concluded that there were no significant benefits to using heavy tamponade instead of conventional SOs in these cases.[29] Double fillings—Double fillings are heterogeneous solutions in which the SFA sinks and the SO floats due to its specific gravity, meaning that they are able to provide superior and inferior tamponades simultaneously. The most commonly used SFA is perfluorohexyloctane (F6H8). The amount of F6H8is much greater than the amount that can be dissolved by SO; therefore, the top part of the bubble consists of SO saturated with dissolved F6H8, whereas the bottom part of the bubble is pure F6H8.The Table 3. Advantages and limitations of the vitreous substitutes used in clinical practice. Advantages Limitations Gases - No need for removal - Non-toxic -Inert - Expansile - Prone positioning after vitrectomy - Expansile gases can produce intraocular pressure elevation (IOP) Silicone Oils - Transparency - Low surface tension - Long-term internal tamponade - Low toxicity - Must be removed within a 6-month period - Optical adjustments may be required - Less effective tamponade of the inferior retina due to its low specific gravity Perfluorocarbon liquids - Clear, colorless and odorless - Similar refractive index to vitreous humour - Stabilization of the retina during vitrectomy - Limited to intraoperative setting - Long-term toxicity - MustbereplacedwithSO Semifluorinated alkanes - Less retinotoxicity than PFCLs due to its lower specific density -SolubleinPFCLsand SOs. - Emulsification - Cataracts Heavy Silicone Oils - Good transparency, high density and viscosity - Good tamponade properties - Less tendency to disperse - Difficult to handle due to its viscosity - Must be removed within a 2-month period. most commonly reported combination is F6H8mixed with 1000 cSt SO in a 3:7 proportion.[83,84] A combined internal tamponade of F6H8and SO may be useful for treating complicated RD with breaks involving the retina’s lower quadrants.[85] In order to clarify all of the advantages and limitations of the vitreous substitutes that are currently used in clinical practice, these characteristics have been included in Table 3. 4. Influence of Vitreous Substitution on Pharmacokinetics of Intravitreal Drugs When the vitreous is substituted with SO, 80% of the vitreous cavity will be filled with the tamponade, while the rest will be replenished with aqueous humour and some vitreous remnants may be present. In the case of inert gas substitutes, the proportion that is to be refilled with the aqueous humour will increase as the gas disappears, and the time it takes to totally disappear will differ depending on the type of gas (Table 1). Artificial substitutes that are currently used in clinical practice differ in terms of their vitreous composition, and this may affect the pharmacokinetics of intravitreally injected drugs.[86] Most of these drugs are water-soluble and will only dissolve in the vitreous aqueous phase. To the best of our knowledge, very few intravitreal pharmacokinetic studies have been conducted on tamponade animal eye models[87,88] in Macromol. Biosci. 2021, 2100066 2100066 (7 of 15) © 2021 The Authors. Macromolecular Bioscience published by Wiley-VCH GmbH www.advancedsciencenews.com www.mbs-journal.de which SO was used as the vitreous substituent. In this sense, Xu et. al investigated the bevacizumab injection (1.25 mg/0.05 mL) in a rabbit model, observing that silicone acted as a temporary depot for controlled drug delivery, delaying drug distribution into the remaining vitreous aqueous phase, where the drug dissolved, before being further distributed into the surrounding tissues. Therefore, it is anticipated that the drug concentration-time profiles will change between the native and filled vitreous. However, given the lack of internal control the intravitreal clearance or halflife differences between SO-vitreous and the control healthy eye were not determined in those studies.[87,88] For ethical reasons it is not possible to perform these types of pharmacokinetic studies in patients. Nevertheless, a few studies have evaluated the safety and efficacy of treatment versus no-treatment in SO-filled eyes with antivirals[89] or bevacizumab injections,[90,91] observing positive clinical outcomes in the treated patients. Several other case reports have shown that the Ozurdex implant (dexamethasone 0.7 mg loaded in a biodegradable sustained-release intravitreal implant (Allergan Inc., Irvine, CA)) appears to be tolerated by and beneficial to patients with SO tamponade.[92–94] However, it must be noted that the reported clinical studies are based on a limited number of patients. The drug release from the implant will depend on the phase in which said implant is located, that is to say the aqueous phase or the SO/gas phase, as this release will only be possible in a medium in which the drug can be dissolved. In the case of acting-gas tamponade, the release may also be dependent on the timing of the gas disappearing. Moreover, the drug release from the implant may be delayed in the filled-eye, and longer drug levels may be maintained, nonetheless, further investigations on an animal models are required.[95] Overall, there is still a lack of quantitative data on the effect of the vitreous substituents on intravitreal pharmacokinetics. Further pharmacokinetic studies must be conducted in order to clarify their effect on the drug concentrations following intravitreal administration, for both drug solutions and implants. 5. Experimental Vitreous Substitutes: Hydrogels There are still numerous inconveniences and limitations to the use of currently available vitreous substitutes in clinical practice. Consequently, the search for new biomaterials that can be used to achieve the ideal vitreous substitute still continues. Previous research attempted to produce vitreous substitutes that boasted similar physiological properties and molecular structure to the vitreous body. The limits of this approach included the toxicity of the compounds and their incapacity to provide sufficient internal tamponade for vitreous replacement surgery.[96] In order to overcome these drawbacks, recent research has focused on developing biocompatible, biodegradable, and injectable hydrogels (natural, synthetic, and smart), which will also act as medium and long-term internal tamponade agents.[2] Hydrogels do not have to be removed after a certain period of time, therefore overcoming one of the main inconveniences related to this procedure. In addition, depending on the types of polymers used for their synthesis, some of their properties can be optimized. Specifically, their viscosity, porosity, good mechanical strength, and the possibility of drug encapsulation make these advantageous for their clinical use in patients. Main hydrogels which have been developed as vitreous substitutes in recent years have been outlined in Table 4. 5.1. Natural Hydrogels The use of HA and collagen as vitreous substitutes has been evaluated due to their great biocompatibility and given that these are the main components of the vitreous. However, they have a poor tamponade effect and a limited retention time in vivo comparing to the results produced by synthetic and smart hydrogels, due to the molecules tendency towards degradation and their low viscosity.[23,97] To increase retention time, HA has been cross-linked through UV and dihydrazide, resulting in biocompatible hydrogels that present good transparency, viscosity, and tamponade effect thanks to their hydrophilic properties, nonetheless, these materials still present relatively short-term stability.[99,103] In addition, cross-linked hyaluronate formulations and combinations of HA with other polymers, such as microbial anionic polysaccharide gellan have also been tested. However, due to the instability of the physical crosslinks, these combinations are not available for long-term use.[97,98,100,101] Aiming to improve this feature, Raia et al. synthesized silk and HA composite hydrogels by cross-linking the tyrosine residues native to silk fibroin and tyramine-conjugated HA. In this sense, the composite silk-HA hydrogel retained the favorable properties of each of the polymers. Consequently, the better control of the water content in the composite matrix exerted by HA and the slow proteolytic degradation of the silk resulted in longer stability and durability.[125] Additionally, Uesugi et al. used a natural polymer, which was not based on collagen or HA as a vitreous substitute. Specifically, they reported the use of PanaceaGel SPG-178 (0.1%), a selfassembling gel, the main component of which is 13 amino acid synthetic peptide. This gel can be injected through a 27-gauge needle and its refractive index, visible light transmission rate, and rheological properties are similar to those of human vitreous. In addition, they carried out a three-month in vivo study in rabbits in which good biocompatibility and no toxicity were observed.[102] Nevertheless, rapid degradation remains a major problem for this type of substitutes, as biomaterials tend to degrade and change their physicomechanical properties in a short period of time. This is a considerable drawback given that the ideal vitreous substitutes must be stable for long periods of time, preferably over three months.[2,6] 5.2. Synthetic Hydrogels Polymeric hydrogels are the next step towards producing the ideal vitreous substitute. These materials are networks of crosslinked hydrophilic polymer chains with extensive swelling, absorbing several times their own weight in water.[126,127] They have a good level of transparency, biocompatibility, and present viscoelastic properties that are similar to the vitreous body, imitating its biofunctionality.[128] Poly(1-vinyl-2-pyrrolidone) (PVP) was the first synthetic polymer to be tested as a potential vitreous substitute. The most commonly reported adverse effects were vitreous opacification and inflammation reaction, resulting in early PVP degradation due to phagocytosis.[129] In addition, 1-vinyl-2-pyrrolidone (VP) monomer was polymerized with divinyl glycol (DVG) as a Macromol. Biosci. 2021, 2100066 2100066 (8 of 15) © 2021 The Authors. Macromolecular Bioscience published by Wiley-VCH GmbH www.advancedsciencenews.com www.mbs-journal.de Table 4. Hydrogels developed as vitreous substitutes and their main characteristics. Hydrogels Polymer content [%] Refractive index Light transmittance [%] In vivo studies Reference Natural polymers Gellan and hyaluronic acid 1 85–95 no [97] Methacrylated gellan gum 1 no [98] Hyaluronic acid 1 1.338 rabbits [99] Hyaluronic acid 3 1.341 rabbits [100] Hyaluronic acid 1–2.2 1.32–1.34 rabbits [101] Peptide gel 0.10 1.3339 96.7 rabbits [102] Hyaluronic acid 1 1.32–1.33 rabbits [103] Hyaluronic acid 1 1.336 75–91 no [23] Synthetic polymers Polyvinyl alcohol methacrylate 9 no [104] Polyvinyl alcohol 7 macaques [105] Polyvinyl alcohol 4 85 no [106] Poly(ethylene glycol) 5 1.339 rabbits [107] Polyvinyl alcohol 5 no [108] Polyvinyl alcohol 1–7 1.3361 93 rabbits [109] Polyvinyl alcohol 4 1.3420 no [110] Acrylic acid and acrylamide 1.25 –1.75 no [111] Poly N-acryloyl glycinamide-polycarboxybetaine acrylamide 1.60 1.3354 93.2 rabbits [112] Smart hydrogels WTG-127 89.3 rabbits [113] Poly(ethylene glycol) 25 1.353 >90 no [114] Poly(ethylene glycol) 10 1.3325 rabbits [115] Sulfobetaine methacrylamide and acryloyl cystamine monomers 5 >90 rabbits [116] Gellan and poly(methacrylamide-co-methacrylate) 0.65–1.29 1.3351–1.3372 87.6–94 rabbits [117] Methacrylic acid, methylacrylamide, and bismethacryloylcystamine 1–1.4 no [118] Poly(ethylene glycol) 0.4–0.7 rabbits [119] Polymethacrylamide and poly-methacrylate 0.9–1.8 1.3345–1.3348 >95 no [120] Hydroxypropyl chitosan and alginate dialdehyde 1–3 1.3348 >80 rabbits [121] Poly(ethylene glycol), poly(propylene glycol), and poly(𝜖-caprolactone) 3–12 1.339–1.344 rabbits [122] Poly(ethylene glycol) methacrylate and poly(ethylene glycol) diacrylate 0.75–5.7 1.3350–1.3359 >90 no [123] Gellan and poly(methacrylamide-co-methacrylate-cobis(methacryloyl)cystamine) 1.3355–1.3370 >83 rabbits [124] cross-linking agent in order to obtain a transparent hydrogel with a similar density and viscosity to the vitreous body.[129] Finally, VP was also co-polymerized with 2-hydroxyethyl methacrylate (HEMA) using diallyl ether (DAE) as a cross-linking agent, resulting in a clear and transparent gel with mechanical properties close to those of the vitreous, however, the main inconvenience was that the elastic properties were reduced or even lost when injected.[130] Polyacrylamide (PAA) has been synthesized by the polymerization of acrylamide, a toxic and carcinogenic substance, with a disulfide cross-linking agent. However, this polymerization process highly improves its biocompatibility. PAA presents good biocompatibility and long-term stability, as well as offering a similar viscosity and density to the vitreous. With regards to adverse reactions, severe ocular inflammation and vitreous opacification have been reported.[131] Poly(2-hydroxyethylacrylate) (PHEA) presented very good physical properties; however, due to the emergence of inflammatory reactions, as well as cataract and glaucoma this substance is no longer being investigated.[4,6] All of the aforementioned polymers presented complications related to inflammation and toxicity. As a result, other polymers such as poly(glyceryl methacrylate) (PGMA) and hydroxypropyl methylcellulose (HPMC) were investigated; however, these did not reach the clinical study stage due to their short degradation time.[132,133] Macromol. Biosci. 2021, 2100066 2100066 (9 of 15) © 2021 The Authors. Macromolecular Bioscience published by Wiley-VCH GmbH