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Review

Biological macromolecules for ophthalmic drug delivery to treat ocular diseases Venkateshwaran Krishnaswami a,c , Ruckmani Kandasamy a,b,c , Shanmugarathinam Alagarsamy a , Rajaguru Palanisamy d , Subramanian Natesan a,b,c,∗ a

Department of Pharmaceutical Technology, University College of Engineering, BIT Campus, Anna University, Tiruchirappalli, Tamilnadu, India National Facility for Drug Development, University College of Engineering, BIT Campus, Anna University, Tiruchirappalli, Tamilnadu, India c National Facility on Bioactive Peptides from Milk, University College of Engineering, BIT Campus, Anna University, Tiruchirappalli, Tamilnadu, India d Department of Biotechnology, University College of Engineering, BIT Campus, Anna University, Tiruchirappalli, Tamilnadu, India b

a r t i c l e

i n f o

Article history: Received 15 November 2017 Received in revised form 10 January 2018 Accepted 17 January 2018 Available online xxx Keywords: Macromolecules Ocular drug delivery Cornea Chitosan Ocular diseases

a b s t r a c t Development of newer drug carrier systems by the researchers has resulted in numerous breakthroughs in the development and manufacturing of ocular products. The ocular bioavailability of drugs at the posterior segment of the eye is a challenging task in the present scenario. Naturally derived macromolecular carriers are widely used to increase the efficacy of ocular drugs. They provide enhanced corneal permeability and retention effect at the surface of cornea for a prolonged period of time. In this regimen the present review focuses towards the major ocular diseases and their prevalence and development of efficient drug carrier systems utilizing various naturally derived macromolecules for improved delivery of drugs to treat ocular diseases. © 2018 Elsevier B.V. All rights reserved.

Contents 1. 2.

3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Ocular diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.1. Age related macular degeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.2. Cataract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.3. Fungal keratitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.4. Retinoblastoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.5. Conjunctivitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.6. Glaucoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.7. Diabetic retinopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Biological macromolecules and drug delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.1. Carbohydrate based macromolecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.2. Chitin based macromolecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.3. Chondroitin based macromolecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.4. Collagen based macromolecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.5. Cellulose based macromolecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Abbreviations: AMD, age related macular degeneration; VEGF, vascular endothelial growth factor; FGF, basic fibroblast growth factor; EGF, epithelial growth factor; RB, retinoblastoma; RGC, retinal ganglion cells; IOP, intra ocular pressure; AH, aqueous humour; ELAM-1, endothelial-leukocyte adhesion molecule; ET, endothelin-1; TM, trabecular meshwork; DR, diabetic retinopathy; HIF-1␣, hypoxia-inducible factor; mTOR, mammalian target of the rapamycin; PEG, polyethylene glycol; CS, chitosan; NPs, nanoparticles; CAP, cellulose acetate phthalate; PLGA, polylactide co-glycolide; RES, resveratrol; QUR, quercetin; 1 O2 , singlet oxygen; HB, hypocrellin B; ROS, reactive oxygen species; A549, human adeno lung carcinoma; h, hours; Ag NPs, nano silver; nm, nanometer; CAP, cellulose acetate phthalate. ∗ Corresponding author at: Room No- 212, Department of Pharmaceutical Technology, University College of Engineering, Bharathidasan Institute of Technology Campus, Anna University, Tiruchirapalli- 620 024, Tamilnadu, India. E-mail address: [email protected] (S. Natesan). https://doi.org/10.1016/j.ijbiomac.2018.01.120 0141-8130/© 2018 Elsevier B.V. All rights reserved.

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3.6. Other macromolecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Introduction Human eye is designed to gather visible information from the surrounding environment. It is segmented into two parts, anterior and posterior pole/segment. Orbit, lids and sclera are the protective structures of the eye. The anterior segment of the eye comprises cornea, aqueous humour, iris, ciliary body and crystalline lens, whereas the posterior segment includes retina and vitreous humour. The optic nerves, optic tracts and visual cortex are the visual signal pathways to the brain. Cornea is the delicate portion of eye and is composed of corneal epithelium, corneal stroma, and corneal endothelium layers. The structural integrity of the cornea is attained by corneal lamellar stroma. The tissue lining the inner surface of the eye and surrounded by vitreous cavity is the retina. The neural retina is composed of neurons, photoreceptors, bipolar cells, horizontal cells, amacrine cells and ganglion cells. Cornea limits the diffusion of most hydrophilic/hydrophobic molecules and lowers the ocular bioavailability (1–7%) [1]. Tear turnover, nasolacrimal drainage, reflex blinking, and ocular static and dynamic barriers are the various anatomical and physiological constraints that limit the corneal drug permeation [2]. The presence of blood retinal barriers makes the systemic administration as an unfavorable approach for delivering drugs to the posterior segment of the eye. Occurrence of several ocular diseases also alters permeation or movement of the drug across the eye. Vision loss is one of the most feared complications of human diseases other than death. Globally ocular diseases affect the patient’s vision and quality of life. World widely an estimate of 285 million people are visually impaired, with an USA scenario of 3.4 million people over the age of 40 years [1]. The vision threatening diseases (Fig. 1) affect both the anterior/posterior segments of the eye which include age related macular degeneration, cataract, keratitis, glaucoma, diabetic retinopathy, retinoblastoma, allergic conjunctivitis and ocular trauma. This review presents prevalence of different vision threatening diseases that are affecting the ocular globe and further focused on drug delivery systems developed using biological macromolecules as a carrier for the delivery of ocular drugs, so that the readers may utilize the information for design and evaluation of drug carrier systems based on macromolecules. 2. Ocular diseases 2.1. Age related macular degeneration Age related macular degeneration (AMD) is a multifactorial degenerative disease affecting the posterior segment of the eye and it is one of the leading causes of blindness in developed countries with progressive loss of central vision in individuals over the age of 50 years. The development of AMD is characterized by abnormal growth of new blood vessels (angiogenesis) in the retinal pigment epithelium which leads to drusen, atropy, and detachment of bruch’s membrane [3]. Still there is no cure for this disease, but the treatments may slow the progression [4]. Among two types of AMD, dry (atropic or nonexudative) and wet (neovascular or emulative), the wet form is characterized with the growth of new blood vessels, leakages of blood and fluid under macula, which leads to consequences of hemorrhage and scar formation (yellow or white spots) in the fundus [4]. Several cellular growth factors such as

vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF) and epithelial growth factor (EGF) are elevated during the retinal angiogenesis due to the abnormalities associated with respective metabolic pathways. Alterations of multiple signaling pathways such as expression of VEGF, matrix metalloproteins, P13K/AKt, and ERK1/2 are gaining importance for controlling retinal angiogenesis. Globally around 8.7% of blindness is caused by AMD [4] and 30 million people are affected by AMD [5,6]. Among these the USA holds the highest risk position (11 millions). In the USA, a large percentage of people of age 50 (1.65 millions) and older are reported to have advanced stages of AMD, and this figure is expected to be doubled by 2020 [5]. The prevalence rate of late AMD in India is comparable to that of western populations. The major risk factors for AMD in India are population aging, and adaptation of western life style and diet [7]. It has been reported that improvement in diet, physical activity, maintenance of blood pressure and avoidance of smoking may decrease the prevalence of the AMD risks. 2.2. Cataract Cataract, the formation of cloudiness/opacification of the crystalline protein in the eye lens, is a major leading cause of blindness globally. The different types of cataracts are cortical, nuclear, or posterior subcapsular. Crystallin (90%) protein is the mature component of lens which maintains the transparency of lens [8,9]. The early onset of cataract is associated with mutations in ␣, ␤ and ␥ crystallin and its associated genes. Pathology of cataracts involves photooxidative stress, non- enzymatic glycation and exposure to hydrophobic drugs which may lead in to high molecular weight aggregates of crystallins and protein insolubilisation with elevated calcium levels in the lens [9,10]. Primarily the reactive hydroxyl radicals are responsible for the oxidative damage of lens. Oxidative stress is also mediated by hyperglycemia induced micro and macro vascular complications [11,12]. Factors such as race, heredity, smoking, UV exposure, nutritional inadequacies, diabetes, and aging lead to cataract formation [13]. Worldwide around 40–60% of the blindness is caused due to cataract and in India cataract is responsible for 50–80% of bilateral blindness. The current treatment options adopted for cataract is the surgical removal of the opaque lens. The current prevalence rate of cataract gets decreased (around 25%) in India due to increase in cataract surgeries [11]. An estimate of 10 year delay in cataract formation may reduce the need of surgery in 50% cases. However the development of a nonsurgical approach for cataract treatment may have beneficial impact on both human health and healthcare costs. In this strategy, multifunctional antioxidants were reported as anti-cataract agents due to their radical scavenging and chelation ability [8]. 2.3. Fungal keratitis Fungal keratitis (keratomycosis) occurs in the inflamed cornea by the attack of fungus such as Candida albicans, Candida glabrata, Candida tropicalis, Candida krusei and Candida parapsilosis [14]. A healthy cornea won’t provide entry for fungus, whereas a traumatic cornea may provide way for fungal pathogens to enter. Fungal keratitis is characterized by corneal ulceration and stromal inflammatory infiltration [15]. The excessive corneal inflammation and

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Fig. 1. Vision threatening ocular diseases associated with the anterior and posterior segments of the eye.

impaired wound healing that occur during fungal keratitis affect miRNA expression [16]. The prevalence of fungal keratitis ranges up to 40% of the microbial keratitis in developing countries such as India and Thailand. In India an estimate of 113 per 100,000 cases are affected by fungal keratitis. Fungal keratitis is usually treated by antifungal eye drops and oral medications. Corneal transplantation may be needed in certain cases where these medicines are ineffective. In some cases, even after corneal surgery vision may not be restored and leads to permanent vision impairment or blindness [17]. 2.4. Retinoblastoma Retinoblastoma (RB) is the malignant solid tumor affecting the retina and commonly occurs among children of less than 5 years old [18]. Although, RB is curable, the untreated versions result in vision loss and ultimately death (99%) [19]. Mutations in the tumor suppressor gene RB1 encoding for retinoblastoma protein, pRb, is responsible for the development of childhood ocular tumor. RB may be unifocal or multifocal. Among the RB affected individuals, nearly 60% have unilateral RB and 40% have bilateral RB. Heritable retinoblastoma occurs due to germline mutations in the RB1 gene or autosomal dominant variant of RB. Individuals with heritable retinoblastoma are also at increased risk of developing non-ocular tumors. Trilateral RB (pineoblastoma), a condition that occurs in 5–15% of patients with heritable form of uni- or bilateral RB which is characterized by the development of an intracranial midline neuroblastic tumor and typically develops between the ages of 20 and 36 months [20]. The treatment options adopted for RB are radiotherapy, cryotherapy, systemic chemotherapy and surgery. The occurrence of RB is estimated to be 1 out of 20,000 live births [19]. In the USA, among children who are diagnosed for RB, about

63% of cancer occurs below two years of age. Occurrence rate is almost equal among male (3.7 per million), female (3.8 per million), whites (3.7 per million) and blacks (4.0 per million). Incidence of RB is age dependent. As age increases, the incidence of both unilateral and bilateral RB decreases [21]. Mean age at diagnosis is 27 months and 15 months in Canada for unilateral RB and bilateral disease, respectively [22]. Drug delivery to treat RB is a challenging task and offers multifactorial difficulties to find a robust and lasting treatment. The widely adopted treatment options for the RB are chemotherapy and radiation therapy. The systemic administration of chemotherapeutic drugs are not able to reach the vitreous body due to blood-retinal barrier. Increasing the dose of drug may endanger into unwanted toxicities. Recent research suggests that release of compensatory proangiogenic factors and angiogenic blood vessels formation is the critical step for the treatment of RB [20]. Even the orbital extension of RB can lead to systemic dissemination through the blood vessels [19]. Since, several signaling pathways are involved in RB treatments addressing multiple pathways may provide efficient therapy. 2.5. Conjunctivitis Inflammation/infection to conjunctival tissue will lead to conjunctivitis. It is the most common ophthalmic disorder, which occurs due to viral, bacterial, fungal infections, allergens and irritants (23) and commonly termed as ‘red eye’ or ‘pink eye’. The causative agents/organisms that are associated with different types of conjunctivitis are bacterial (Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Chlamydia trachomatis and Neisseria gonorrhoeae) viral (adeno viruses), allergic (allergens, pollens) and chemical (fumes, dust, chemicals)

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conjunctivitis. Hyperemia, edema and ocular discharge mark the characteristic features of conjunctivitis arising due to the dilatation of the bulbar and palpebral conjunctiva blood vessels [24]. The symptoms associated with conjunctivitis include redness in the white part of the eye, increased tears, burning eyes, blurred vision and increased sensitivity to light. Based on the causes, conjunctivitis can be classified into infectious and noninfectious. Most common infectious causes are bacteria and viruses and noninfectious causes are allergens and irritants. Conjunctivitis affects all genders, ages and races [23]. Among the infectious conjunctivitis cases worldwide, viral conjunctivitis are responsible for 15–70%, bacterial conjunctivitis accounts for 42–80% and 3% is contributed by chlamydial conjunctivitis. Nearly 40% of global population is suffering from allergic conjunctivitis [25]. Antiviral drugs (acyclovir, ganciclovir) were widely used for viral conjunctivitis. It has been reported that lower vitreal level of dipeptide monoester prodrugs of ganciclovir was observed in case of conscious animals compared to anaesthetized animal [26]. 2.6. Glaucoma Glaucoma (both open angle and closed angle) is a well-known ocular disease characterized by an optic neuropathy which causes slow degeneration of optic nerve axons and death of retinal ganglion cells (RGC) leading to complete blindness [27]. It is generally associated with increase in intraocular pressure (IOP) due to abnormal production or blockage of the aqueous humour (AH). The increased IOP may damage the optic nerve. The symptoms of glaucoma begins with a gradual loss of vision progress in to complete loss of vision at final stage, and it is irreversible. It is estimated that by 2020, up to 80 million people will be affected by glaucoma. Women patients form 55% of open angle glaucoma, 70% of angle closure glaucoma and 59% of all forms of glaucoma in 2010. It is estimated that Asian population have 47% of glaucoma and 87% of those with angle closure glaucoma [28]. The IOP elevation is maintained by balancing the production of the aqueous humour fluid in the ciliary body, along with its outflow through the posterior chamber of trabecular and the uveoscleral pathways. Glaucoma is classified into open angle glaucoma and angle closure glaucoma. Open angle glaucoma is an asymptomatic, progressive optic neuropathy characterized by enlarging optic disc cupping and visual field, which leads to increased resistance to aqueous outflow through the trabecular meshwork. Angle closure glaucoma is a condition in which the pressure developed inside the eye becomes too high due to the obstruction in the drainage pathways [27]. Drugs used for the glaucomatous patients aids to regulate the aqueous humour production or outflow. Risk factors for glaucoma development include age, race, diabetes, family history, myopia, pseudoexfoliation, migraine and caliber of retinal vasculature [28]. Multiple factors acting either on cell bodies or their axons are responsible for RGC death rather than any one of them functioning individually [29]. Oxidative stress plays a major role in glaucomatous neurodegeneration [30]. The antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase are present in AH. Ascorbic acid is present in higher concentrations in vitreous humour, cornea, lachrymal film, central corneal epithelium and AH. The glutathione system protects the eye tissues from the damage caused by low concentrations of hydrogen peroxide. These antioxidant levels in AH decrease with aging that causes elevated IOP [31]. In addition to oxidative process, evidence for nitrative processes are also found in glaucoma. It has been reported that nitric oxide may be an important mediator in RGC death in glaucoma. Altered oxidant/antioxidant balance in glaucoma patient’s eye may result in a number of molecular changes that contribute to the development of this disease [32].

The close connection between vascular function and oxidative damage is also related to the physiological activities of the endothelium. The endothelium is involved in the modulation of vascular permeability through the release of endothelin-1 (ET) and nitric oxide [33]. ET is the most potent vasoactive peptide present in ocular tissues and plays a potential role in the development of glaucoma. Increased ET levels in the trabecular meshwork (TM) cells causes TM channel contraction that reduces the AH outflow. In addition, ET evokes apoptosis of RGC by reducing the blood flow to the RGC [34]. The pathogenic role of oxidative stress in open angle glaucoma is further supported by the increased expression of the endothelial-leukocyte adhesion molecule (ELAM-1) in the TM of glaucoma patients [35]. ELAM-1 expression is controlled by the autocrine feedback mechanism of activation of interleukin-1 through the nuclear transcription factor NFkB, and provides protection against oxidative stress [36]. 2.7. Diabetic retinopathy Diabetic retinopathy (DR) both proliferative and nonproliferative, is a highly specific vascular complication of both type I and type II diabetes mellitus, DR is elicited due to chronic exposure to high blood glucose, which ultimately leads to the alteration of the blood retinal with a thickened basement membrane and ends with progressive damage to the retina. Proliferative DR is initiated by hypoxia associated with the formation of newer blood vessels (angiogenesis). Pathogenesis of DR is characterized by the formation of leaky, primitive and disorganized vascular networks. Changes in microvascular structures of retinal tissues occur through the formation of advanced glycation end products in chronic hyperglycemic conditions and results in weakening and blockage of blood vessels. Lack of perfusion activates hypoxiainducible factor (HIF-1␣) through PI3K/AKT signaling pathway, which upregulates VEGF leading to the uncontrolled and unorganized growth of new vessels, increased permeability/breakdown of the blood retinal barrier, accelerated loss of retinal neurons through apoptotic cell death and exudates formation. Vitrectomy, a surgical procedure, is used in the case of advanced PDR, wherein vitreous gel and blood from leaking vessels in the back of the eye are removed to focus light rays properly on the retina. But this surgery offers only a temporary relief and does not stop further leaking of blood. Alternatively, corticosteroid medications are administered as intra vitreous injections to reduce swelling of the macula, slowdown the vision loss and improving vision. Sustained release of corticosteroids from implants can improve the vision by interfering the mechanisms that cause inflammation and damage of the blood vessels. Current treatment with antibody based anti-VEGF agents (Ranibizumab and Aflibercept) targets the expression of VEGF and thereby decrease the leakage, reduce the edema and offer improved vision and lower risk of developing other eye complications [37]. The mammalian target of the rapamycin (mTOR) in PI3K/AKT/m-TOR signaling pathway is a serine-threonine kinase that forms two distinct protein complexes, mTOR complex 1 and 2 (mTORC1 & C2) in association with other proteins. mTOR is hyperactivated during various types of angiogenic conditions including DR and may lead into activation of a broad spectrum of cellular mechanisms including survival, proliferation, growth, metabolism and angiogenesis [38]. Recently mTOR inhibitors such as everolimus, sirolimus, tacrolimus are suggested for the treatment of DR. The prevalence of DR is strongly related with respect to diabetes [39]. After 20 years of diabetes, nearly all patients with type 1 diabetes and above 60% of patients with type 2 diabetes have some degree of retinopathy. Current global estimate of diabetic patients with DR is 92.6 million, including 17.2 million with proliferative DR, 20.6 million with diabetic macular edema and a total of 28.4

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million people with DR that is considered to be a threat for vision. Microvascular complication which affects the peripheral retina, the macula or both leads to visual disability and blindness in people with diabetes. The risk for DR development can be reduced by early detection, timely management of blood glucose and blood pressure. Oxidative stress and inflammation due to the upregulation of proinflammatory cytokines caused by hyperglycemic conditions are considered major factors for the development of DR. Laser photocoagulation, vitrectomy and pharmacological therapies are adopted to treat sight threatening retinopathy. Laser photocoagulation helps to stabilize the condition by sealing off/shrinking the leaky blood vessels and potentially prevents vision loss. However, this procedure is unlikely to improve the vision and comes with a risk of laser scar. 3. Biological macromolecules and drug delivery Macromolecules are larger molecules possessing higher molecular weight, generally obtained by polymerization of smaller units of monomers. The commonly utilized macromolecules in drug delivery include nucleic acids, proteins, carbohydrates, polyphenols and lipids. Nucleic acids and proteins are linear polymers having specific sequence and fixed molecular weights with variations in chain length and lack of polydispersity. On the other hand, natural polysaccharides are having broader polydispersities, branched molecular structure and various reactive sites on glycoside units [40]. Polysaccharides are considered to be very similar to synthetic polymers due to these structural properties and make them as promising candidates for the development of drug carrier systems. Various biological macromolecules such as carbohydrates, chitin/chitosan, chondroitin and derived macromolecules that are utilized for delivering ocular drugs are discussed below. The biological macromolecules for ocular drug delivery should be selected based upon the stability, biodistribution of macromolecular agent, degradation, clearance by the mononuclear phagocytes of the reticuloendothelial system and its ability to penetrate into the target tissue [30,41]. 3.1. Carbohydrate based macromolecules Carbohydrates are naturally acquired biocompatible polymers and widely utilized in drug delivery. These polymers are possessing the prerequisites of biocompatibility, biodegradability, hydrophilicity, stability, non-toxicity and adhesive nature that make them to be chosen as one of the best natural polymers by ocular researchers. Polysaccharide based polymers, both positively (chitosan) and negatively (alginate, heparin, pectin) charged, posses the intrinsic capability to recognize specific cells with targeting moieties through specific receptors [42]. Carbohydrate based polymers can be modified specifically as per the requirements due to the presence of various (hydroxyl, amino, carboxyl) reactive groups. Alginate is a naturally derived polysaccharide consisting of (1–4)-linked ␤-d-mannuronic acid and (1–4)-linked ␣-l-guluronic acid isolated from brown sea algae (Macrocystis pyrifera, Laminaria hyperborean and Ascophyllum nodosum) [43]. Commercially, alginate is obtained from sea weeds by microbial fermentation and extraction process. Its relative properties of biocompatibility, hydrophilicity and in situ gelling property make alginate as a suitable polymer candidate for ocular drug delivery. Alginates form hydrogels with certain divalent cations [43]. Modifications are done in the alginate and used in thiolated alginate-albumin nanoparticles, alginate poloxamer microparticles, hydrated thiolated alginate, alginate-poly (lactic-co-glycolic acid) nano/micro

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hydrogel matrices, chitosan calcium alginate microspheres, alginate modified by microenvironmental interaction with calcium ion and polyethylene glycol-anthracene modified alginate [41]. Depending upon the physicochemical properties such as viscosity inducing property, sol-gel transition, and water-uptake ability alginates are available as M- block and G- blocks. The gelling property of alginate gets induced by complexation with divalent ions and at decreased pH conditions [44]. Wong et al. developed collagen alginate composite gel (0.7%) based encapsulated cell therapy using GDNF-secreting HEK293 cells for the posterior segment ocular diseases [45] and to provide a sustainable GDNF delivery thereby to overcome the repeated invasive intravitreal injections and to improve the ocular bioavailability at posterior pole (retina). Alginate concentration and initial cell density are the important parameters in the optimization of encapsulated cell therapy [45]. Alginate based microparticulate ocular drug delivery system was developed by Batyrbekov et al. using two anti-cancer drugs cyclophosphane and 5-fluorouracil for the treatment of retinoblastoma. These particles showed 5- 8 times enhanced anti-cancer activity in comparison to that of the free drugs [46,47]. Mittal et al. developed an in situ delivery system for the treatment of glaucoma using pectine alone or in combination with sodium alginate. They observed that ion activated in situ gelling ocular systems undergo phase transition in the presence of cations (present in the tear fluid), thus may supports for the long term residence of drug in the cornea [48]. 3.2. Chitin based macromolecules Polysaccharides are designated into linear or branched chain depending upon the nature of the monosaccharide unit, which includes varying number of functional groups such as hydroxyl, amino, and carboxyl groups for chemical modification. Chitin is a natural polysaccharide derived from the exoskeleton of crustaceans such as crabs and shrimps, in addition chitin also present in the cell walls of fungi and bacteria. Chitosan is a copolymer of ␤-(1–4)-linked d-glucosamine and N-acetyl-d-glucosamine. Chitosan is a biodegradable, non-toxic, mucoadhesive and biocompatible carbohydrate based natural polymer obtained during deacetylation of chitin (obtained from shrimps and crabs) and widely utilized in ocular drug delivery. Various modifications done in chitosan were carboxy methylation, carboxy ethylation, reductive amination with phosphorylcholine glyceraldehydes, sulfation, N- or O-acylation alkylation and quaternarization. Chitosan has the unique property of mucoadhesion due to surface positive charge which facilitates adherence to mucosal surfaces (mostly negative charged) and allows it to reside on the ocular surface for prolonged time after administration. Besides the above mentioned advantages, chitosan appears to enhance the permeability of the drugs across the cornea transiently due to the interaction with the tight junctions located between epithelial cells. The mucoadhesive nature of macromolecules upon ocular surface during topical application of eye drops is shown in Fig. 2. Due to the mucoadhesive property chitosan is utilized to enhance the ocular bioavailability of both hydrophilic and lipophilic drugs. Chitosan stabilized nanoparticles posses the characteristics of controlled drug release, which improves drug solubility, stability, and efficacy, and reduces toxicity. Higher molecular weight and higher deacetylated chitosan reported to exhibits improved permeability effect [49]. The water insolubility and highly viscous characteristics of unmodified forms of chitosan holds the tendency to coagulate with proteins at high pH values [50]. Chitosan is positively charged in dilute acids [51,52]. Franca et al. developed bimatoprost-loaded ocular inserts using chitosan by solvent casting method for the treatment of glaucoma [49] and found to lower

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Fig. 3. Sol-to-gel transition characteristics of chitosan on exposure to pH variation.

Fig. 2. Mucoadhesion characteristics of chitosan.

intra ocular pressure for a consecutive period of 4 weeks [49]. Cyclosporine-loaded chitosan nanoparticles provided an enhanced level of delivery to the cornea and conjunctiva (10 folds) when compared to cyclosporine suspension [53]. Chitosan can be combined with other permeation enhancers in order to elicit a synergistic or additive effect. Recently chondroitin sulphate-chitosan nanoparticles was developed for the ocular delivery of bromfenac sodium and to improve the corneal permeation and retention time. Higher transcorneal permeation (1.62-fold) and corneal retention (1.92-folds) of bromfenac were observed when compared to marketed eye drops [54]. Rajendran et al. developed acyclovir loaded chitosan nanoparticles using ionic gelation method for the management of viral infections in eye. They developed the nanoparticles at the size of 200–495 nm with spherical smooth morphology with a zeta potential range of +36.7 to +42.3 mV. The encapsulation efficiency and loading capacity of the developed formulations were around 56–80% and 10–25% respectively. Acyclovir loaded chitosan nanoparticles showed the enhancement of corneal permeation, contact time and bioavailability of acyclovir for the treatment of ocular viral infections [55]. Daptomycin loaded nanoparticles were developed by Silva et al. for the treatment of bacterial endophthalmitis in eye with an approach to improve the effectiveness of treatment and reducing systemic toxicity. They prepared particles with the size of 200 nm and low polydispersity index, and positive zeta potential, with an encapsulation efficiency of 80–97%. The antimicrobial activity of daptomycin gets preserved when daptomycin was encapsulated into chitosan nanoparticles. This may be due to the binding capacity of chitosan to the negatively charged bacterial cells which may and sustains the antibacterial effect for a prolonged period of time [56]. Chitosan was utilized to develop temperature dependent insitu gelling systems for the efficient delivery of drugs to the eye [57]. In-situ gelling systems are liquid polymer solutions that upon topical administration in to ocular cul-de-sac undergo phase transition of liquid to viscoelastic gel in response to environmental conditions. Phase transitions may be induced or triggered by environmental stimuli such as temperature, pH, ions, solvent and light. It is well documented that improved bioavailability can be obtained by increasing the viscosity of drug formulation in the precorneal region, due to slower drainage from the cornea. The pH responsive characteristics of chitosan may be due to the protonation/deprotonation of amine in chitosan at altered pH conditions which results in hydrophilic/hydrophobic nature to induce sol/gel transistions [58]. The pH dependent sol-to-gel transition characteristics of chitosan is shown in Fig. 3.

Temperature and pH triggered novel in situ gelling system using poloxamer and chitosan of Timolol maleate was reported by Gupta et al. [59]. The developed system was a clear solution that converted into gel at temperatures above 35 ◦ C and showed improved residence time. Several in situ gelling systems using chitosan as viscosity modifier were reported to enhance residence time of various drugs such as ciprofloxacin [59], gatifloxacin sesquihydrate [60] and pilocarpine [61]. The improved delivery and synergistic intra ocular reducing effect of quercetin and resveratrol-loaded polyethylene glycol (PEG) modified chitosan (CS) nanoparticles for the treatment of glaucoma was developed by Subramanian et al. [62] using ionic gelation method. The obtained nanoparticles were spherical shaped at a size range of around 100 nm with amorphous nature. Incorporation of PEG in the formulation resulted in intermolecular hydrogen bonding between the amino hydrogen of chitosan and the oxygen atom of PEG forming a CS-PEG semi-interpenetrating network due to the electropositive and electronegative effects, thus improves the biocompatibility and stability of the formulation in physiological fluids. Permeation of resveratrol across the excised rabbit cornea from the quercetin- and resveratrol-loaded nanoparticles was observed to be 78%, which is higher than that of resveratrol loaded PEG modified CS NPs (52%), which may be due to decreased level of p-glycoprotein inhibition exhibited by the presence of quercetin in the formulation (Fig. 5). Reduced IOP with nanoformulation was sustained for 8 h. Peak IOP (5.5 ± 0.5 mmHg) reducing effect of resveratrol and quercetin loaded PEG modified CS NPs (0.5%) was observed at 4 h and it is higher than that of reduced IOP (4.8 ± 0.5 mmHg) shown by resveratrol loaded PEG modified CS NPs. Similarly higher concentration of resveratrol and quercetin loaded PEG modified CS NPs (1% and 1.5%) showed sustained IOP reducing effect, for up to 8 h (Fig. 4). In this formulation resveratrol gets embedded/crosslinked in the polymer matrix, The prolonged effect of the formulation may occur due to increased mucoadhesion of chitosan in the formulation that may interact with mucin effectively and thereby induces the synergestic effect [62]. 3.3. Chondroitin based macromolecules Chondroitin sulphate is a naturally existing mucopolysaccharide based water soluble cationic polymer and a component of human body. Chondroitin sulphate composed of repeated disaccharide units of ␤-1, 4-linked D-glucuronic acid and ␤-1, 3-linked N-acetylgalactosamine. Chondroitin sulphate is widely utilized for colon targeted delivery [61]. Due to its non-toxic, non-irritant, mucoadhesive and biodegradable properties it is also used as a polymer in ocular drug delivery. Bromfenac loaded chondroitin sulphate- chitosan nanoparticles developed by Tara et al. were found to be at size range of 245.6 ± 14.22 nm, with polydispersity index of 0.187 ± 0.016, holding zeta potential of +37.59 ± 4.05 mV

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Fig. 4. IOP lowering effect of topically applied various concentrations of RES-loaded PEG modified CS NPs (A), RES and QUR loaded PEG modified CS NPs (B) in normotensive rabbits.

Fig. 5. Nanoparticulate systems of photosensitizer loaded PLGA nanoparticles, high resolution TEM images of nanoparticles at different magnifications (A), In vitro drug release of photosensitizer loaded PLGA nanoparticles (B) Phototoxicity effect of photosensitizer loaded PLGA nanoparticles at 2 h light irradiation (C) and Singlet oxygen generation efficiency of photosensitizer loaded PLGA nanoparticles (D), HB- Hypocrellin B, AgNPs- nano silver, HB NPs- HB loaded PLGA nanoparticles, HB Ag NPs- HB and nano silver loaded PLGA nanoparticles (datas previously published).

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Table 1 Biological macromolecules based ocular drug delivery formulations. S No

Category

Macromolecule

Formulation with incorporated drug

Therapeutic activity

References

1

Carbohydrate

Alginate

Ocular gels (Levofloxacin)

[45]

2

Carbohydrate

Alginate

[46]

3 4

Carbohydrate Polysaccharide

Sodium alginate Chitosan

Glaucoma Glaucoma

[47] [48]

5

Polysaccharide

Chitosan

Dry eye syndrome

[50]

6 7 8

Polysaccharide Polysaccharide Mucopolysaccharide

Chitosan Chitosan Chondroitin sulphate

Fibroblasts

Collagen

10

Polysaccharide

Cellulose

Nanoformulations (Ibuprofen)

To improve corneal permeation Ocular viral infections Improved corneal permeation and retention effect Ocular residence, to prolong the drug activity Improves viscosity/corneal residence time/pH responsive behaviour

[53] [54] [52]

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Microparticles (Cyclophosphane and 5-fluorouracil) In situ gel (5-fluorouracil) Ocular inserts (Bimatoprost loaded) Nanoparticles (Cyclosporine loaded) Bromofenac sodium Nanoparticles (Acyclovir) Nanoparticles (Bromofenac sodium) Corneal shields

Posterior segment ocular diseases Retinoblastoma

and entrapment of 71.72 ± 4.43%. They observed a biphasic release pattern of initial burst release (24.23 ± 1.82%) for 1 h followed by sustained release profile (94.36 ± 6.36%) for 24 h. Corneal uptake of drug was confirmed by confocal laser scanning microscopy and it showed improved corneal permeation of 44.79 ± 2.54 ␮g/cm2 /h. Increase in the corneal permeation of drug might be due to the ionic interaction between protonated amino groups of nanoparticles with the negatively charged mucin. Further the ocular tolerability of the developed formulation was proved in chick chorio allantoic membrane [63].

3.4. Collagen based macromolecules Collagen (composed of fibroblasts) is a type of structural protein abundant in most connective tissues. In mammals collagen based tissues are present in bones, tendons, corneas, cartilage, blood vessels, gut, intervertebral discs, dentin and cartilages. Collagen based corneal shields possessing high oxygen permeability is widely used to treat corneal epithelial damages; the dissolution rate of collagen shields upon corneal surface depends upon the rate of collagen crosslinking. Ofloxacin-loaded corneal shields have been reported to possess improved delivery of drug for corneal disorders [64]. Collagen is used in ocular drug delivery systems due to its ability to increase the ocular residence time, to prolong the drug activity, to target intraocular tissues, to improve shelf life, to lower the visual side effects and there by achieving higher bioavailability [64].

3.5. Cellulose based macromolecules Cellulose are basically odorless biodegradable polysaccharides composed of a linear chain of ␤(1 → 4) linked D-glucose units, insoluble in water and most organic solvents. Cellulose in the form of methyl cellulose, cellulose acetate phthalate and hydroxyl propyl methyl cellulose is widely utilized in ocular drug delivery. These macromolecules are reported to elicit pH sensitive pattern. These molecules coagulates at the ocular pH that makes to release the drug in a sustained manner over the corneal surface. Cellulose based macromolecules may improves the viscosity of formulations, thereby enhances the corneal residence time. Mohan et al. [65] developed ciprofloxacin loaded in situ gelling system using poly acrylic acid (Carbopol 940) as the gelling agent in combination of viscosity-enhancing agent (hydroxy propyl methylcellulose) and pluronic F-127 as the thermal reversible gelling agent.

[64] [65]

The dual stimuli responsive system consist of pH responsive Ibuprofen loaded cellulose acetate phthalate (CAP) nanoparticles dispersed in temperature responsive gel forming solution containing Pluronic F127 and F68 was reported by Subramanian et al. [66]. The nanoparticles dispersed in Pluronics solution (21% F127 and 10% F68) showed good phase transition behaviour at ocular temperature and moderate viscosity that makes them suitable for ocular administration. The sizes of the nanoparticles were ranged between 159–356 nm. The in vitro drug release study showed about 70% of drug release in 8 h. The prolonged retention of gel consistency and controlled corneal permeability of the drug (180 g in 2 h) from the system in response to the pH and temperature was also reported [66]. The biological macromolecules based ocular drug delivery formulations are shown in Table 1. 3.6. Other macromolecules Polylactide co-glycolide (PLGA) is a USFDA and European Medicine Agency approved biocompatible and biodegradable macromolecule based polymer available in the form of microspheres used in drug delivery. It provides tremendous application in targeting, imaging, diagnostics, therapy and in the development of biocompatible devices/tissue scaffolds. Various combinations of lactic acid and glycolic acid present in PLGA different forms (25:75, 50:50 and 75:25%) are available in three different sizes of 75 ␮m, 100 ␮m and 120 ␮m. The release/biodegradation of PLGA depends upon pH and temperature in the biological systems, but the PLGA microspheres will hold 2- 4 months to degrade completely in aqueous system due to the presence of PLGA ester linkage. The presence of 0.005% (sodium dodecyl sulphate) may improve the suspendability of PLGA microspheres in aqueous environment. The morphological changes including surface erosion, geometry changes and loss in molecular weight/mass will occurs in the biodegradation process. PLGA gained significant interest for the delivery of lipophilic/hydrophilic drugs (Ma et al. [67]). Recently, PLGA loaded nanoparticles based ocular drug delivery systems were reported for sparfloxacin [68]. PLGA nanoparticles possess negative charge, and can be made positive/neutral charged by surface modifications. The metabolic hydrolysis of PLGA yields non- toxic oligomer and monomer of lactic and glycolic acids. The ratio of lactic acid and glycolic acid in PLGA plolymer plays a vital role in manipulating the degradation rate of PLGA from days to months and afford a controlled/sustained release pattern in ocular delivery [69]. PLGA nanoparticles functions as a diagnostic and

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imaging agents. Surface modification enables the targeting ligand attachment for its site specific drug delivery [70]. Release of the drug molecule from the PLGA matrix is governed by osmotic pumping, diffusion through water filled pores, diffusion through the polymer and erosion (no drug transport). The degradation of PLGA, both in vivo and in vitro, depends upon the method of preparation, physico chemical parameters (pH, temperature and ionic strength of environment), size, shape, morphology and intrinstic properties of the polymer. With the drug loaded PLGA nanoparticles after entering in to the cells through receptor mediated endocytosis, charge reversal takes place in acidified endosomes and destabilization of endolysosomal membrane causes the release of drug molecules from the nanoparticles. Even the intracellular uptake of nanoparticles is unaffected by serum. PLGA nanoparticles possess cationic charge inside the endosomes [71], which reduces the chance of toxicity commonly associated with cationic lipids/polymers. The application, mechanism and targetability of PLGA polymer to deliver various anti-cancer drugs like dexamethasone, paclitaxel, vincristine sulphate, curcumin, campothecin, doxorubicin, cisplatin, etoposide and rapamycin in the microparticulate/nanoparticulate formulations was illustrated by Acharya et al. [72]. They demonstrated that PLGA also functions as a diagnostic agent and MRI contrast agent for imaging. The polyethylene glycol modified PLGA nanoparticles provides a higher circulation time in blood due to presence of PEG chains. The PLGA nanoparticles reduces cellular toxicity by getting internalized at the lysosomal and endosomal compartments of the cell, without destabilizing the lysosomes. Nanoparticulate system utilizing photodynamic therapy with an objective to achieve enhanced production of singlet oxygen (1 O2 ) was reported recently by Subramanian et al. [73]. This formulation was developed using hypocrellin B as the photosensitizer in combination with nano silver with a derived PLGA polymer for the treatment of posterior segment eye diseases like age related macular degeneration. The optimized spherical nanoparticles contained 2.60 ± 0.06 mg/mL of hypocrellin B with a size range of 135.6–828.2 nm and were negatively charged with a narrow polydispersity index. The average encapsulation of 92.9 ± 1.79% was achieved; the drug release from the PLGA polymeric nanoparticles followed a biphasic pattern with an initial burst of 3.50% during first 8 h along with a sustained release pattern of 47.82% within 3 days. Generation of reactive oxygen species (ROS) by the formulation was significantly higher in nano silver incorporated formulation along with photosensitizer. The formulations showed a concentration and time dependent phototoxicity to A549 (human adeno lung carcinoma) cells in the presence of light providing a superior phototoxic effect (82.2% at 50 ␮M) at 2 h irradiation (Fig. 5). The ocular biodistribution studies revealed that intravenous administration of these PLGA based formulation lead into significant exposure to the posterior segment of the eye.

4. Conclusion Biological macromolecules are paving greater role in ocular drug delivery due to their property to enhance the corneal permeation, capability for improving the corneal residence, enhancement in the viscosity of formulations, pH and temperature responsive characteristics of gel in to sol transformation, decreasing the clearance of topically applied ocular formulations through tears, improving the interaction with ocular mucins and improving the ocular bioavailability and distribution at the posterior segment of eye for various ocular disorders. Therefore developing formulations utilizing biological macromolecules coupled with innovative recent technologies may offer superior beneficial therapeutic effects.

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Acknowledgements Authors gratefully acknowledge the financial support received from Department of Biotechnology, New Delhi, Govt. of India under the project BT/PR3804/MED/32/220/2011 and Department of Science and Technology, New Delhi, Govt. of India supported National Facility on Bioactive Peptides from Milk (VI- D&P/545/201617/TDT).

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