Vitreous Humor: Composition, Characteristics and Implication on Intravitreal Drug Delivery

Abstract

Purpose: Intravitreal administration of drug molecules is one of the most common routes for treating posterior segment eye diseases. However, the properties of vitreous humour changes with the time. A number of ocular complications such as liquefaction of the vitreous humour, solidification of the vitreous humour in the central vitreous cavity and detachment of the limiting membrane due to the shrinking of vitreous humour are some of the factors that can drastically affect the efficacy of therapeutics delivered via intravitreal route. Although significant research has been conducted for studying the properties of vitreous humour and its changes during the ageing process, there have been limited work to understand the effect of these changes on therapeutic efficacy of intravitreal drug delivery systems. Therefore, in this review we discussed both the coomposition and characteristics of the vitreous humour, and their subsequent influence on intravitreal drug delivery.

Methods: Articles were searched on Scopus, PubMed and Web of Science up to March 2022.

Results: In this review, we discussed the biological composition and biomechanical properties of vitreous humour, methods to study the properties of vitreous humour and the changes in these properties and their relevance in ocular drug delivery field, with the aim to provide a useful insight into these aspects which can aid the process of development of novel intravitreal drug delivery systems.

Conclusions: The composition and characteristics of the vitreous humour, and how these change during natural aging processes, directly influence intravitreal drug delivery. This review therefore highlights the importance of understanding the properties of the vitreous and identifies the need to achieve greater understanding of how changing properties of the vitreous affect the therapeutic efficacy of drugs administered for the treatment of posterior eye diseases.

Keywords:

• Ocular drug delivery systems
• intravitreal therapy
• biomechanics
• posterior segment drug delivery

Introduction

The eye is the delicate sensory organ of human body, which is involved in sending visual sensory information to brain via the optic nerve. The pathophysiology associated with the various ocular diseases is unique due to structural intricacies of the eye. Eye diseases are economic burden on patient in addition to deteriorating health. As the leading causes of blindness globally, the most prevalent posterior segment ocular diseases are age-related macular degeneration (AMD), diabetic retinopathy (DR) and diabetic macular edema (DME). With approximately 4.01% population suffering from AMD and 2.99% suffering from DR followed by 1.56% people suffering from glaucoma.¹ AMD and DR are neurogenerative in nature with DR being microvascular in nature affecting the peripheral vision and AMD is a macrovascular in nature with the choroidal neovascularisation near the macula affecting the central vision in patients. Altered permeability of blood vessels and capillaries in DR leads to decrease in oxygen supply causing intraretinal neovascularisation, followed by vitreous haemorrhage and subretinal fibrosis.² Pathophysiologically, AMD is multifaceted disorder in which deposition of drusen in Bruch’s membrane causes oxidative stress in retinal photoreceptor cells. Drusen is defined as accumulation of lipofuscin - a yellowish to dark brown pigment, as described in the year 1842 by Hannover,³ along with proteins, sugars and cell debris from RPE cells.⁴ Accumulation of drusen is a ‘hallmark’ of dry AMD. Approximately 80% of the AMD cases are dry AMD, as the age progresses further, the accumulation of drusen leads to oxidative stress in RPE leads to the formation of leaky immature capillaries in choroid region near macula, causing central vision loss.

Currently available treatment methods to treat AMD, DR and DME are monthly or bimonthly intravitreal (IVT) injections of anti-VEGFs. Ranibizumab (Lucentis®, Genentech) an anti-VEGF antibody fragment in the concentration of 6 mg/ml and 10 mg/ml aqueous solutions (pH 5.5) is widely used for management of AMD, macular oedema following retinal vein occlusion (RVO), DME, DR and myopic choroidal neovascularisation (mCNV).⁵ Ranibizumab is a fragment of full-length anti-body Bevacizumab (Avastin^®, Genentech) available off-label for the management of AMD and DR. Bevacizumab is a ∼150 kDa anti-VEGF agent works by neutralising VEGF-A and preventing non-selective binding to VEGF-1 and VEGF-2. Bi-monthly injections of Aflibercept (Eylea^®, Regeneron, 40 mg/ml) a recombinant fusion protein has been found to be as effective as monthly injections of Lucentis^® (0.5 mg/injection).⁶

Brolucizumab (Beovu^®, Novartis) a VEGF inhibitor with recommended dose of 6 mg monthly for initial three doses and every 8–12 week thereafter for treatment of wet AMD. Brolucizumab is the smallest single chain antibody fragment with molecular weight nearly 26 kDa. Similar to bevacizumab and ranibizumab, brolucizumab binds with three isoforms of VEGF-A (VEGF₁₁₀, VEGF₁₂₁ and VEGF₁₆₅). Pegaptinib sodium (Macugen^®, Eyetech Pharmaceuticals Inc. and Pfizer Inc.) at a 0.3 mg/90 µl concentration is also indicated for the treatment of wet AMD and administered every six weeks. Pegaptanib sodium is an oligonucleotide with 28 amino acid and approximately 50 kDa molecular weight. A study on 147 patients with wet AMD suggested a the longer circulation time of pegaptanib in eyes along with the major elimination of drug happening via systemic circulation was likely a result of pegylation side chains on a 28 nucleotide sequence 7).

Uveitis, an anti-inflammatory disease of the uvea, the layers of the eye underneath the sclera (ciliary body, iris, and choroid), requires the long-term intravitreal injection of anti-inflammatory agents. Triamcinolone is a synthetic glucocorticoid with anti-inflammatory properties and injected through intravitreal injections to patients unresponsive to topical treatment. A suspension is available in two strengths 40 mg/ml (Kenalog^®, Bristol-Myers Squibb Company) and 80 mg/ml (Trivaris^®, Allergan).⁷ Trivaris^® is an 80 mg/ml triamcinolone acetonide suspension for intravitreal injection for the treatment of uveitis. Whereas Kenalog^® is an injectable TA suspension for intramuscular and intra-articular injection for the ocular diseases sympathetic ophthalmia, temporal arteritis and uveitis. Ocriplasmin (JETREA^®, Inceptua) a recombinant form of human plasmin approved in the year 2012 for the intravitreal treatment of vitreomacular adhesion. However, the use of Ocriplasmin has been discontinued due to the observed adverse side effects such as vitreous floaters, eye pain post injection and photopsia.

As the posterior segment ocular diseases are complex, multifaceted, and blinding, they also need long-term drug treatment to maintain or improve the best corrective visual acuity (BCVA). Long-acting sustained release formulations are desired to reduce the economic burden on patients and could help in controlling the rising clinical demand. The development of long-acting sustainable drug delivery system could also aid in preventing the common side effects of these monthly injections, which are vitreal detachment, patient discomfort, and endophthalmitis. Table 1 shows clinically approved sustained release formulation for management of different ocular disorders. Intravitreal implants of anti-inflammatory drugs for the long-term sustained drug release are under clinical trials. Ozurdex^® (Allergan) is a dexamethasone implant by Allergan Inc. with 0.46 mm diameter and 6 mm length. It is a biodegradable implant made up of PLGA can maintain therapeutic concentration of dexamethasone for up to 6 months with initial loading dose for 2 months followed by 4 months of plateau release phase.¹⁴ Iluvien^® (Alimera Sciences, Inc.) is a fluocinolone acetonide non-biodegradable intravitreal implant made up of polyvinyl alcohol and silicone indicated for the treatment of DME. Iluvien^® implant containing 190 µg of fluocinolone acetonide and it can release 0.25 µg drug per day for up to 36 months. Retisert^® (Bausch Lomb) and Yutiq^TM (EyePoint Pharmaceuticals) are another non-biodegradable intravitreal implants of fluocinolone acetonide for the treatment of uveitis. Retisert^® was developed for the treatment of chronic non-infectious uveitis. Retisert^® implant is a fluocinolone acetonide tablet with silicone elastomer cup reservoir with release orifice and a polyvinyl alcohol membrane. The major drawback of Retisert^® implant is that it needs to be sutured into posterior segment of the eye through pars plana. Yutiq^® is a non-bioerodible intravitreal implant able to release drug for 36 months at a rate of 0.25 µg/day. Yutiq^® is a preformed implant with 0.18 µg fluocinolone acetonide and injected using 25 gauge needle via intravitreal injection.

Table 1. List of clinically used sustained release intravitreal implants – recreated with permission from Parenky et al.⁸ Reproduced from 1. Parenky AC, Wadhwa S, Chen HH, Bhalla AS, Graham KS, Shameem M. Container Closure and Delivery Considerations for Intravitreal Drug Administration. AAPS PharmSciTech (2021) 22: Licensed under CC BY 4.0.

Product name	Molecule	Owner/company	Dose/duration	Needle gauge	Dimensions	References
ILUVIEN^®	Fluocinolone acetonide	Alimera Sciences, Inc.	0.19 mg	25 gauge	3.5 mm × 0.37 mm	^9
OZURDEX^®	Dexamethasone	Allergan, Inc.	0.7 mg	22 gauge	0.46 mm × 6 mm	^10
RETISERT^®	Fluocinolone acetonide	Bausch & Lomb Inc.	0.59 mg/30 months	NA (sutured)	3 mm × 2 mm × 5 mm	^11
YUTIQ^™	Fluocinolone acetonide	EyePoint Pharmaceuticals US, Inc.	0.18 mg	25 gauge	3.5 mm × 0.37 mm	^12
Port delivery system	Ranibizumab	Roche	0.5 mg/monthly	Refillable implant (sutured)	Size of a rice grain	^13

Port delivery system (PDS) (Susvimo™) by Genentech is the first and one of its kind ophthalmic implant for continuous delivery of anti-VEGF (ranibizumab) for wet AMD for up to 6 months and putatively reducing treatment burden on patients with posterior segment eye diseases. PDS is a refillable ocular implant designed to be sutured onto sclera of patients near pars plana and nearly the size of a rice grain.¹⁵ Intravitreal in-situ forming microparticles of PLGA with surface modification loaded with Sunitinib was developed by Graybug Vision, Inc. (Redwood City, CA, USA). The technology allows delivery of drugs to both anterior and posterior segment of the eye without significant initial burst release and can deliver drugs from days up to several months. Clinical trials in wet AMD patients with minimum three prior anti-VEGF intravitreal injections with 0.25, 0.5, 1, and 2 mg of sunitinib-loaded microparticles reported improvement in BCVA. Data of Phase 2 clinical trials revealed that patients on 1 mg Sunitinib did not require supportive therapy for the six months.¹⁶

Anti-VEGF has become the first line of treatment for AMD, DR and DME. However, 8 out of 10 patients are unresponsive to IVT of anti-VEGF injections, despite their excellent activity in-vitro, some patients do not respond, or only respond partially. Additionally, frequent dosing is required as the large anti-bodies have low ocular half-life. The suboptimal response to anti-VEGF therapy could be attributed to changes in vitreous humour with aging and higher inter-patient variability.¹⁷

Composition and functions of vitreous humor

Occupying the vitreous cavity, the vitreous humour is a homogenous, viscoelastic hydrogel structure that helps to maintain the shape of the eyeball through its continuous contact with the retina by inner limiting membrane (Figure 1(A)). The vitreous also acts to prevent physical damage to lens and retina by absorbing any mechanical impact, as well as preventing oxidative damage.^20,21 Another crucial role of the vitreous is in the transmission of light, as the transparent nature of the vitreous allows the passage of refracted light towards the photoreceptors of the retinal tissue. The received light is then converted to electrical signals by the photoreceptors which are subsequently processed in the brain. Similar the cornea, the vitreous has a refractive index of 1.336,^22,23 which is crucial to its role in the transmission of light.

Figure 1. (A) Human vitreous of a 9-month-old child dissected of the sclera, choroid, and retina, is still attached to the anterior segment, Reprinted by permission from Springer: Eye,¹⁸ Imaging vitreous. Sebag et al. (2002), (B) Orientation of collagen fibres in the vitreous humour, reprinted by permission from (Springer), Eye,¹⁹ adult vitreous structure and postnatal changes. Le Goff MM, Bishop PN. Eye (2008) 22.

The vitreous is composed of two phases, namely the liquid phase and gel phase, which exhibits non-Newtonian properties.²⁴ The main component of the vitreous is water (>90%), however it also possesses solid fibrillar components in the form of glycosaminoglycans and collagen (Figure 2). Collagen fibres are formed through three polypeptide α chains, of which glycine is present in every third residue, which then form a triple helix structure. Other common amino acids used in this formation include hydroxyproline and proline, which impact additional hydrogen bonds within the structure to stabilise the triple helix formation.¹⁹

Figure 2. Schematic showing the key components of the vitreous humour.

Within the vitreous, collagen exists in the vitreous at an approximate concentration ranging between 280 and 1,360 µg/ml in regular arrangement. Of the 27 collagen types which exist, either in fibril or sheet conformations, the most common collagen type within the vitreous humour is type II (60–75%).²⁵ Type II formation occurs following the enzymatic cleavage of soluble procollagen by N- and C-propeptide to form the type II polypeptides which then form fibrils.²⁶ Other collagen types present at lower concentrations include type V and XI collagen (10–25%),¹⁹ which alongside type II form heterotypic fibrils whereby a fibril core composed of type V and type XI collagen is surrounded by type II collagen.²⁷ The network created by the formation of these collagen fibrils plays an essential role in maintaining the overall shape of the vitreous (Figure 1(B)), as well as imparting important properties such as flexibility. The density of the fibre networks increases around the periphery of the vitreous humour so as to generate both anterior and posterior hyaloid membranes which keep the vitreous separate from the rest of the eye.²⁴ Additionally, immune modulating cells known as hyalocytes reside in the vitreous humour periphery where they comprise the acellular region of the vitreous and are key in modulating inflammation responses in diseases such as diabetic retinopathy.²⁸

Along with the collagen components of the vitreous, non-collagenous components such as glycoproteins, i.e. fibrillin and opticin, are also essential to vitreous properties and function. While fibrillin acts a key component of zonules,²⁹ opticin is present on collagen fibril surfaces and is suggested to have anti-angiogenic functions.³⁰ Additionally, extended chains of disaccharide units known as glycosaminoglycans are also present in the vitreous. Glycosaminoglycans undergo modification (e.g. acetylation, sulphation, etc.) e.g. heparin undergoes sulphation to allow attachment to serine residues of a core protein. Furthermore, with the exception to hyaluronic acid, all glycosaminoglycans combine to a protein core to form structures known as proteoglycans.

Hyaluronic acid, an anionic polymer, is the predominant glycosaminoglycan present in the vitreous at a concentration of approximately 140–340 μg/ml.²⁷ However, the reported values vary in literature. The viscoelastic nature of the vitreous is generated through the presence of negatively-charged, hydrophilic hyaluronic acid molecules surrounding the collagen fibril network, which helps to generate swelling, pressure and bring stability to the surrounding proteoglycan and water molecules.²⁰ Unlike other glycosaminoglycans, hyaluronic acid is not attached to a core protein and will not undergo modification processes such as sulfation.³¹ The concentration of hyaluronic acid within the vitreous varies, with highest concentrations present in the posterior region of the vitreous cortex (Figure 3).³³

Figure 3. Schematic showing the distribution of hyaluronic acid concentrations present in the vitreous humour, reprinted from Elsevier: pharmacokinetic aspects of retinal drug delivery,³² del Amo EM, et al, 134–185, Copyright (2017), with permission from Elsevier.

Hyaluronic acid is vulnerable to enzymatic degradation by hyaluronidases,³⁴ although little is currently known about this process and the subsequent implications on drug delivery.

The vitreous is also known to contain non-structural proteins, such as immunoglobulins and complement proteins. In diseased conditions such as macular degeneration, which affects both the vitreous and retina, an increase in the protein concentration within the vitreous may be observed. Loukovaara et al. (2015) determined the protein concentration to significantly increase in the vitreous of a patient with diabetic retinopathy versus healthy vitreous i.e. 5.1 ± 1.8 mg/ml versus 4.7 ± 1.2 mg/ml.³⁵ Furthermore, under disease states, such as in diabetic retinopathy, the presence of unique proteins such as enolase and catalase has been observed.³³

Other glycosaminoglycans present include chondroitin sulfate and heparin sulfate, however these are present at much lower concentrations combined with trace levels of salts.

Chondrotin sulfate combines with core proteins to form two types of proteoglycans. The first, a large proteoglycan, of which isoforms are involved in cell adhesion and regulation for example,³⁶ is known as versican. The second form is type IX collagen, which is composed of three differing polypeptide chains. Heparin sulphate meanwhile is a predominant component of the inner limiting lamina, as well as other basement membranes and has been implicated in several diseases of the eye, including AMD.³⁷

Studies investigating the biomechanical and rheological properties of the vitreous humour are limited in literature. It is important to understand these mechanical properties of the vitreous and how they are impacted with aging (e.g. liquefaction) and disease status. Additionally, in the case of retinal detachment, the replacement of the vitreous with a suitable substitute requires adequate knowledge of vitreous properties.

In young healthy individuals, the kinematic viscosity of the vitreous humour has been reported to be between 300 and 2000 cSt and is higher in the central vitreous versus the periphery.³³ However, with aging the vitreous humour undergoes a natural and irreversible process known as liquefaction which results in a decrease of its viscosity. This is thought to be a result of enzymatic degradation of proteoglycans, resulting in collagen aggregation. The subsequent retraction of the gel-like vitreous leaves spaces within the vitreous known as lacunae, which become filled with liquid.³⁸ Often this process results in complete posterior vitreous detachment, or vitreomacular traction, whereby the vitreous partially detaches but remains in contact with the macula. The degradation of the vitreous can also result in the development of cataracts,³⁹ through the process of protein aggregation within the lens.

The changes in the diffusion properties of the vitreous humour have been studied by Bilgil et al. (2011) The authors used apparent diffusion coefficient as a marker for vitreous viscosity measured using magnetic resonance imaging. It was found that there was a drastic change in the diffusion coefficient within the paediatric and aged population. Although there was the difference observed between various aging groups but a general trend of increased diffusion with the increasing age was established (Figure 4(A–C)). The authors also studied the diffusion weighted imaging to characterise the aqueous movement within the vitreous, which showed non-significant change within aged groups. This change in the diffusion coefficient of the vitreous could be attributed to relation of the collagen and hyaluronic acid matrix.⁴⁰

Figure 4. (A) MRI scan of Infant Eye for measuring the diffusivity i.e. ADC. (B) MRI scan of aged eye for measuring the diffusivity. (C) Graph depicting the increasing in the molecular diffusivity of vitreous humour with aging. Used with permission of American Society of Neuroradiology from diffusion changes in the vitreous humor of the eye during aging, İ Meral and Y. Bilgil, 32(8) 2011; permission conveyed through Copyright Clearance Center, Inc. (D) Simulation of the aqueous humor flow in anterior and posterior segment of eye. (E) Simulation of the distribution of Anti-VEGF drug in eye following Intravitreal injection. Reproduced from 1. Dörsam S, Olkhovskiy V, Friedmann E. Modeling and Simulation of the Aqueous Humor Flow in the Human Eye. PAMM (2019) 19:e201900462. Licensed under CC BY 4.0.

Kasdrof et al. (2015) compared the diffusion of polystyrene and liposomal nanoparticles by estimating the diffusing mobility by single particle tracking study. It was observed that light reflection of the particles exhibited defusing behaviour and charge played important role in the particle mobility. It was also observed that the molecular degradation of hyaluronic acid increase in the fraction of mobile particles indicating that enhanced diffusivity. This can be correlated with the age-related liquefaction of vitreous humour.⁴¹

Significant amount of research has suggested that the movement of the aqueous humour from the posterior segment of eye through the vitreous humour exiting via retinal pigmental epithelium. Smith et al. (2020) provided strong evidence of aqueous humour outflow from the vitreous humour by considering various parameters such as the movement of radioactive material inside and its clearance from anterior segment of eye. This movement of aqueous humour could contribute to the vitreous humour flow dynamics and how drug distribution takes place within vitreous humour.⁴² Based on the theory of aqueous humor flow across the vitreous Dorsam et al. (2019) performed the simulation of the distribution of anti-VEGF drug in the vitreous cavity (Figure 4(D,E)). The researchers concluded that the distribution of drug with size smaller than the vitreal pore is dependent upon charge, site of injection and aqueous flow dynamics.⁴³

Additionally, the process of liquefaction has important implications for drug delivery since viscosity is one key mechanical property of the vitreous that will have significant influence on drug diffusion, for example. The process of liquefaction has been shown to result in a slight increase in the diffusivity of molecules through the vitreous due to the movement of the vitreous viscosity similar to water. Similarly, for more complex sustained drug release systems, e.g. cationic liposomes, have also been shown to experience increased diffusivity through the vitreous following liquefaction.³³

To the best of our knowledge, the only study investigating how mechanical properties are implicated with age was conducted in bovine eyes in 2015, finding both storage modulus and loss modulus values to decrease with age versus younger eyes, however this difference was not found to be significant.⁴⁴ The elastic modulus of the vitreous has also been shown to vary between species, with the vitreous of pig eyes showing a much higher modulus (31.2 ± 12.2 Pa) that those of human eyes (1.17 ± 0.56 Pa).⁴⁵

Techniques for the characterisation of vitreous humor viscosity

Viscosity

Viscosity is one of the key parameters of the vitreous humour that aids to the shock absorbing properties hence protecting the lens and retina from mechanical damage. The vitreous dehydration as well as liquefaction that are known to be age-related changes of vitreous that can be effectively understood by observing the viscoelastic properties of vitreous humor. Different methods to study viscosity of vitreous humour are listed in Table 2. Several studies have been performed to study the biorheology of vitreous humour. Shear rheometry is one of the most widely used technique. Recently, Tram et al. (2018) used Kinexus ultra + rheometer for studying the viscoelastic properties of vitreous humour. The solid as well as liquid components of vitreous were studied and the study concluded that over time the vitreous gel becomes stiffer in nature, whereas the vitreous liquefied. (Figure 5(A–C)) The simultaneous processes of vitreous stiffening and liquefaction could lead to alteration of stress distribution within ocular tissue and result into development of retinal tears and macular holes.²¹

Figure 5. (A) Intact human vitreous humour attached to the lens and the iris. (B) Experimental setup used for performing the rheometry of vitreous humor. (C) Amplitude sweep result of solid phase of human vitreous humor sample. Reproduced 1. Tram NK, Swindle-Reilly KE. Rheological properties and age-related changes of the human vitreous humor. Front Bioeng Biotechnol (2018) 6:199. Licensed under CC BY 4.0. (D) Ultrasonograph of healthy eye showing no visible echodensities. (E) Demarcation of whole-central vitreous ROI outlined in red in posterior segment of eye from ultrasonography. (F) Demarcation of whole-posterior vitreous ROI outlined in green in posterior segment of eye from ultrasonography. Used with permission of Association for Research in Vision & Ophthalmology (ARVO), from Mamou J, Wa CA, Yee KMP, Silverman RH, Ketterling JA, Sadun AA, Sebag J. Ultrasound-Based Quantification of Vitreous Floaters Correlates with Contrast Sensitivity and Quality of Life. Invest Ophthalmol Vis Sci, 56, (2015); permission conveyed through Copyright Clearance Center, Inc.

Table 2. Methods and techniques used to study the properties of vitreous humour.

Properties of vitreous humour	techniques used	Reference
Viscosity	Shear rheometry	Tram et al.^21
	Micro rheometry	Lee et al.^46
	Periodic oscillations	Weber et al.^47
Appearance and visualization	Dark field slit microscopy, clinical slit lamp bio microscopy, standard and scanning laser ophthalmoscopy, ultrasonography, optical coherence tomography, magnetic resonance and Raman spectroscopy, and dynamic light scattering	Sebag J^18

In-vitro imaging of vitreous humour

Although quite rare, in vitro imaging of eye can provide various intricate details about such as degradation and status of collagenous matrix of collagen and its precipitation that contributes to the change of vitreous properties. A number of techniques utilised for the in vitro assessment of vitreous humour are explained below.

Light microscopy

Light microscopy can be effective tool for in vitro assessment of the vitreous humor, as it can be used to observe and differentiate between peripheral liquefied vitreous and the centrally located solidifying vitreous. Los et al (2003) performed a study in in vitro human eyes to assess the process of synchysis (liquefaction of vitreous) and syneresis (solidification of vitreous humour) upon senescence/aging.⁴⁸

Transmission electron microscopy (TEM)

TEM is an imaging technique based upon the diffraction of the electron beam and could be used to resolve the structure of the matrix in 3 dimensions. TEM could be used to visualize the collagenous and proteoglycan matrix present in the vitreous humour. TEM could also be used for assessing the degradation of proteoglycans. In one of the studies it was observed that the chain length of the collagen fibres in the liquefied vitreous is shortened, leading to loss of viscoelastic properties of the vitreous and often resulting to posterior vitreous detachment.⁴⁸

Clinical slit lamp bio microscopy

Clinical slit lamp biomicroscopy is one of the most commonly used procedures for ocular examinations. A typical biomicroscopy set up consists of a light slit and binocular microscope which are parfocal (same focal length) which could be adjusted to observe the area of interest in eye. Biomicroscopy has been widely used for the examination of cataracts, corneal injury, scleral damage, macular degeneration, and presence of foreign bodies within eye, as well as vitreous haemorrhages and abnormalities.⁴⁹

Standard and scanning laser ophthalmoscopy

Scanning laser ophthalmoscopy is a variant of another clinical slit lamp biomicroscopy where the biomicroscopy is modified in order to observe the fluorescence within the ocular tissue. Hence it could be used for observation of living tissue such as retina and RPE. The diffusion of different molecules can also be studied inside the eye using this technique. Basile et al. (2011) estimated the concentration of NIR die following an intravitreal injection using confocal scanning laser ophthalmoscopy. The study also yielded the information about the diffusion of dye within the vitreous cavity which could a great tool for studying the pharmacokinetic distribution and clearance of drugs within vitreous humour.⁵⁰

Ultrasonography

Ultrasonography is a diagnostic procedure which is based upon the change in diffraction of sound waves by the difference of density between various organs. It has been widely used for diagnosis of retinal detachment, vicious haemorrhage, and vitreous detachment. Ultrasonography suffers from limitation of low resolution however, it is sensitive enough to differentiate between retinal detachment, vitreous detachment and vitreous haemorrhage, all of which require immediate clinical intervention (Figure 5(D,E)).⁵¹

Further, quantitative ultrasonography (QUS) can also be used for estimation of floaters present within the vitreous humour that are formed due to the degradation of collagen fibrils in vitreous humour. It is observed that vitreous floaters can affect the contrast sensitivity of patience and hence QUS could be a great technique for the quantification of floaters).⁵²

Optical coherence tomography (OCT)

OCT is one of the most widely used techniques for ocular diagnosis it is based upon the principle of interference of low intensity light and it can be used to visualise the cross sections of ocular tissue with non-contact setup. OCT is widely used for visualisation of membranous tissue such as cornea, sclera, retina, retinal pigmental epithelium, Bruch’s membrane and inner limiting membrane. Hence, it could be a useful tool for visualisation of vitreous detachment as well as detachment of inner limiting membrane due to changes in vitreous humour.⁵³

Magnetic resonance imaging (MRI)

MRI is a technique used for visualisation of biological tissue as a result of its interaction with magnetic field. As most of the biological tissues are made up of water, they can be easily visualised using MRI. Ocular MRI is a great tool to estimate the dimension of different ocular tissues and vitreous humour which are majorly affected in diseased condition such as cataract. The resolution of MRI imaging can also be improved by administration of contrast agent which aid in better visualisation of tissue. Further, MRI imaging can also be used for different type of functional visualisation of ocular tissue such as blood flow dynamics and metabolism with use of specific reagents.⁵⁴

Raman spectroscopy

Raman spectroscopy is widely used for determination of the molecular composition of matter. Raman spectroscopy is widely used for qualitative studies of ocular lenses.⁵⁵ However, it has been also used for estimating the materialistic properties of vitreous humour in one of the reports by Sebag et al. (1999) it was absorbed that the vitreous obtained from patients post vitrectomy show prominent peaks at 1604^-cm and 3057^-cm. Such peaks were observed in the patient suffering from diabetic retinopathy.⁵⁶

Dynamic light scattering (DLS)

DLS is a technique based upon the diffraction of light by nanoparticulate materials. It is widely used for studying the particle size and distribution within different kind of matrixes. Hence, it could be very useful techniques for studying the vitreous floaters as well as degraded collagen protein fibrils formed inside the vitreous humour due to ageing. And study performed by Sebag et al (2002) compared in vitro as well as non-invasive DLS technique to study the effect of cross linking and aggregation of collagen fibrils. The authors concluded that DLS could be effectively used for protection and quantification of early molecular effects of collagen degradation and aggregation inside the vitreous humour.¹⁸

Effect of vitreous humour changes on intravitreal drug delivery

The pharmacodynamic efficacy of intravitreal therapy is dependent upon the molecular diffusion of the therapeutic molecule across the vitreous humour and passing across barriers such inner limiting membrane. However, due to aging, the properties of vitreous humour drastically change, for e.g. it is observed that the viscosity of vitreous humour in the periphery is drastically decreased due to the liquefaction whereas the central vitreous humour tends to solidify over the period of time. This changes in the properties of vitreous humour can lead to unpredictable therapeutic efficacy. Furthermore, certain diseased conditions such as AMD and DME can lead to detachment of inner limiting membrane, vitreal haemorrhage and changes in the thickness of retina, as well as formation of drusen which can also affect the pharmacokinetics of drug following an intravitreal injection.

Diffusion of small and large molecules in a viscous medium like vitreous humor has been studied in detail. Highly concentrated protein solutions often show higher viscosity due to formation of protein clusters within the solution and this may lead to decreased translational diffusion of the protein.⁵⁷ This if taken in perspective of intravitreal injections of anti-VEGF agents could present a challenging scenario. The monthly injection of anti-VEGF agents is done from highly concentrated solutions with concentrations of up to 25 mg/ml. This high local concentration of protein could promote clumping and delay the diffusion. The other factors that could affect the diffusion of macromolecules are charge, size and hydrophilic interaction of macromolecules within vitreous humor. In one instance, Käsdorf et al. (2015) showed that the nanoparticles often show decreased diffusion in the vitreous humour when compared with diffusion in water. It was observed that majority fraction of nanoparticles (up to 75%) showed slow and delayed diffusion in vitreous humour and only up to 25% showed particle diffusion similar to water by single particle tracking studies conducted using florescent microscopy. It was also observed that PEGylation of nanoparticles enhanced the diffusion fraction by up to 50%.⁴¹

Studies by Kim et al. (2005) based upon the diffusion of MRI agent from intravitreal implant across the vitreous humor concluded that although there is a simultaneous drug diffusion of MRI agent taking place from implant there could be substantial differences in concentration of drug at different location due to differential movement as well as location of implantation, hence demanding better modelling as well as implant placement strategies for uni-positional implants.³²

One of the physiological condition that could severely impact diffusion of molecules in vitreous humor is liquefaction. The age-related degradation of vitreous humour can also cause the liquefaction of vitreous that is known as syneresis. The research suggests that approximately 20% of vitreous humor exists in liquid state by the age of 14–18 years, which tends to grow up to 50% by the age of 80–90 years. The syneresis is governed by the aggregation of collagen fibrils present in the solid vitreous that leads to redistribution of collagen content in the eye. The other hypothesis suggests that degradation of collagen fibrils b chondroitin ABC lyase can also lead to aggregation of collagen fibrils and further progression of syneresis. This degradation and redistribution of collagen is a major reason for the posterior vitreous detachment (PVD). PVD is a gradual process of splitting away of the cortical vitreous gel from ILL on the inner surface of the retina. It is a common occurrence and can affect up to 25% population. The PVD progression is gradual process and can range from early PVD, i.e. early stages of vitreous liquefaction and weakening of post-basal vitreoretinal adhesion, to complete PVD, where the vitreous is completed detached from the inner limiting membrane and the retinal layers.¹⁹ (Figure 6)

Figure 6. Schematic representation of different stages of posterior vitreous detachment in aged individuals. Reprinted by permission from (Springer), Eye,¹⁹ Adult vitreous structure and postnatal changes. Le Goff MM, Bishop PN. Eye (2008) 22.

The viscous matrix of vitreous humour is loosely packed and has a mesh size of approximately 500 nm in bovine eyes. Even the liquid state of vitreous that is prominently present in aged eyes do not pose a significant diffusion barrier. The vitreous humour does not pose as significant barrier for soluble molecules as well as particles of size below 500 nm. However, diffusion is significantly reduces for positively charged molecules as well as microparticles. Along with diffusion, the vitreal convection also plays significant role in pharmacokinetics of sustained release ocular formulation. However, it was reported that the vitreous convention flow as negligible effect on the drug distribution for soluble intravitreal drugs.³³

Since drug diffusion occurs through the vitreous humour and towards the inner limiting membrane, followed by movement to the retinal layer, PVD could have very detrimental effect on the bioavailability of drugs aimed for the treatment of posterior eye disease. Importantly, the effect of PVD on the treatment of anti-VEGF did not show any significant differences when compared with the normal patients.⁵⁸

Conclusion

In recent years, there have been significant advances in the treatment of posterior eye diseases, such as AMD and diabetic retinopathy, through the use of small molecules and biologics which are delivered both as liquid formulations or formulated into sustained release intravitreal implants. These therapeutics are commonly administered directly into the vitreous humour through intravitreal injection where they move towards the target site, typically the retina, primarily via diffusion. The properties of the vitreous are therefore essential to achieving the desired therapeutic efficacy, given their influence over drug diffusion. Over time, the properties of the vitreous humour are known to change through processes such as liquefaction, which has the potential to decrease the efficacy of such systems. This review therefore highlights the importance of understanding the properties of the vitreous and identifies the need to achieve greater understanding of how changing properties of the vitreous affect the therapeutic efficacy of drugs administered for the treatment of posterior eye diseases.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

DM and SG are funded by the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie Actions [grant agreement – No 813440]