Recent applications of PLGA based nanostructures in drug delivery

Accepted Manuscript Title: RECENT APPLICATIONS OF PLGA BASED NANOSTRUCTURES IN DRUG DELIVERY Authors: Maria Mir, Naveed Ahmed, Asim ur Rehman PII: DOI: Reference:

S0927-7765(17)30450-2 http://dx.doi.org/doi:10.1016/j.colsurfb.2017.07.038 COLSUB 8701

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

20-4-2017 6-7-2017 16-7-2017

Please cite this article as: Maria Mir, Naveed Ahmed, Asim ur Rehman, RECENT APPLICATIONS OF PLGA BASED NANOSTRUCTURES IN DRUG DELIVERY, Colloids and Surfaces B: Biointerfaceshttp://dx.doi.org/10.1016/j.colsurfb.2017.07.038 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

RECENT APPLICATIONS OF PLGA BASED NANOSTRUCTURES IN DRUG DELIVERY Maria Mir, Naveed Ahmed, Asim.ur.Rehman* Department of Pharmacy, Quaid.i.Azam University, Islamabad Pakistan 45320 *

Corresponding Author

Asim.ur.Rehman Department of Pharmacy Quaid.i.Azam University Islamabad, Pakistan 45320 Tel: +923229821867 Email: [email protected] Graphical Abstract

Highlights:     

1. Degradation rate of PLGA greatly influences the release kinetics of drugs. 2. Proper adjustment of factors affecting degradation is required to get efficient DDS. 3. PLGA nanostructures are suitable for molecular level modifications for targeting. 4. PLGA NPs have revolutionized the in-vivo molecular imaging and therapeutic monitoring. 5. Multifunctional PLGA NS resulted in enhanced drug delivery to cancer, CVDs, CNS etc.

Abstract Over the years, issues associated with non-biodegradable polymers have paved the way for biodegradable polymers in the domain of pharmaceutical and biomedical sciences. Poly (lacticco-glycolic acid) (PLGA) is considered one of the most thrivingly synthesized biodegradable polymers. To formulate polymeric nanostructures, PLGA has attained noteworthy attention due to its controllable properties, complete biodegradability and biocompatibility, well defined formulation techniques and easy processing. This review focuses on fabrication techniques of PLGA based nanostructures and their advanced biomedical applications covering drug delivery and in-vivo imaging. Researchers have extensively investigated the potential of PLGA nanoparticles for target specific and controlled delivery of various micro and macromolecules including drugs, peptides, proteins, monoclonal antibodies, growth factors and DNA in multifarious biomedical applications like neurodegenerative and cardiovascular diseases, inflammatory disorders, cancer and other dreadful health disorders. Beside this, PLGA is being employed for theranostic purposes where polymer is attached with contrast agents for imagingdirected chemo or photo thermal combinative therapy. Multifunctional PLGA nanostructures have given an avenue to future nanomedicine to consider simultaneous drug delivery, molecular imaging, and real-time monitoring of therapeutic response. This review describes the applications of PLGA in drug delivery revealing its current progress and direction for future research.

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Keywords: Biodegradable polymer; polymeric nano-composites; targeted drug delivery; theranostics.

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Graphical abstract

PLGA based nanostructures for drug delivery. Figure illustrates the salient features of PLGA (i.e., biocompatibility, biodegradability, mechanical strength and easier to modify) owing to which this polymer is being used for effectual designing of a wide variety of nanostructures including polymeric micelles, dendrimers, nano capsules, nanoghosts, nanogels and theranostic agents.

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Table of contents: 1. 2. 3. 4. 5.

Introduction Synthesis of PLGA Properties of PLGA Biosafety of PLGA Polymer biodegradation 5.1 Factors affecting biodegradation 6. PLGA-based nanostructures and their formulation techniques 7. Applications of PLGA based nanostructures in drug delivery 7.1 Role of PLGA in drug delivery to CVDs 7.2 Applications of PLGA to treat inflammatory diseases 7.3 Applications of PLGA to treat infections 7.4 Applications of PLGA in drug delivery to CNS 7.5 Cancer therapy with PLGA based nanostructures 7.5.1. PLGA based nanostructures for anticancer drug delivery 7.5.2. PLGA based gene delivery to treat cancer 7.6. PLGA in theranostics 8. PLGA nanotechnology based products in clinical trials and market 9. Conclusion and future prospective 1. Introduction In the field of drug designing, synthesis and discovery of novel therapeutic agents with improved biological activities has been the primary focus of researchers. Although, this is still fundamental field of research, but now more attention is being given to the delivery approaches through which these drugs are administered. Encapsulation of drugs in polymers is one of these attractive approaches for drug delivery [1]. Through the decades, issues related to non-dissolvable polymers like, polyacrylates, polysiloxanes or polypropylene have paved the way for natural and synthetic biomaterials in the field of pharmaceutical and biomedical sciences. These biomaterials are capable of in-vivo degradation, either through enzymes or by non-enzymatic hydrolysis or both, to give biocompatible and non-toxic by-products. Elimination of these by-products occurs through normal physiological pathways. Biomaterials which are being used for drug delivery can be categorized mainly in two classes i.e. naturally occurring polymers and synthetic biodegradable polymers. Naturally occurring polymers include inorganics (hydroxyapatite) and complex sugars like chitosan and hyaluronan while synthetic polymers include comparatively hydrophobic materials like α-hydroxy acids (a class that contains polyanhydrides, poly lacticco-glycolic acid (PLGA) and others) [2]. Use of biopolymers is increasing due to growing awareness in consumers about environment safety and global warming. Biopolymers have less harmful effects on environment in comparison with commodity plastics based on fossil fuels. These can be recovered from agricultural feedstock, microbes and marine fauna and waste of these industries can also be employed for biopolymer’s production [3]. 4

Desired outcomes of any medical therapy do not rely only on the pharmacokinetic and dynamic profile of the therapeutic agent, but more importantly on its bioavailability in humans at the desired site of action which in turn depends upon delivery approach [4, 5]. Nanostructures (NS) based carriers are capable of revolutionizing disease therapy through temporally controlled drug delivery [6]. Controlled release of toxic drugs selectively at target sites results in lower side effects, reduced doses, increased compliance and ultimately improved quality of patient’s life [7]. Polymeric nano-particles (PNPs) are optimal carriers for drug delivery due to their tunable characteristics. Many efforts have been done to render them suitable for in-vitro and in-vivo applications. Their physical and biochemical properties, like size, nature of the surface, and the polymer types were studied and synthetic methods were adjusted to obtain good qualities [8, 9]. Various methods for synthesis of PNPs have been adopted according to the nature of agent to be entrapped and its final application. Biodegradable PNPs have been preferred for nano encapsulation of variety of bioactive molecules including drugs, proteins, DNA and imaging agents due to their controlled release property, biocompatibility, subcellular size, stability in blood, non-inflammatory and non-immunogenic nature [10, 11]. Multifarious synthetic polymers have been used for making PNPs. All the materials are not suitable for nanomedical applications, as they are required to be biocompatible by Food and Drug Administration (FDA). Additionally, suitable biodegradation kinetics, good toxicological profile, drug loading efficacy and good mechanical properties are also important requirements for polymer selection [12]. Synthetic biodegradable polymers including polyamides, polyesters and polyurethanes have been used increasingly due to their controllable properties in terms of molecular weight and shape (linear, branched, dendritic, etc.). Amongst all biomaterials, poly lactic-co-glycolic acid (PLGA) emerged as the most promising material showing great potential to be used as carrier in drug delivery and as scaffolds in tissue engineering. PLGA NS have great potential for targeting, imaging, and therapy [13, 14]. PLGA has a prolonged history in medicine as material for biodegradable sutures [15, 16]. Major advantage of PLGA is that, it undergoes complete biodegradation in an aqueous medium [17]. PLGA has gained significant attention among the different polymers developed for formulation of polymeric NS, owing to its salient features including (i) biocompatibility and biodegradability, (ii) approval from Food and Drug Administration (FDA) and European Medicine Agency for drug delivery systems, (iii) feasibility to design sustained release, (iv) practicable surface modifications for provision of stealthness and effective biological interactions [14, 18, 19] (v) protection of drug from degradation, (vi) well described preparations and synthesis approaches in accordance with various types of drugs e.g. hydrophilic or hydrophobic small molecules or macromolecules, (vii) possibility to target specific organs or cells [20]. This review explains the rationale for using PLGA as carrier for drug delivery and discusses adjustable properties and factors that can affect characteristics of these drug delivery systems. Fabrication techniques for development of PLGA based nanostructures have also been explained. This review also explores the applications of these nano polymeric systems for drug delivery in various dreadful diseases including cardiovascular, neurodegenerative, infectious, inflammatory and more importantly in cancer and theranostics. It reveals that how PLGA based nanostructures have revolutionized the drug delivery. 5

2. Synthesis of PLGA PLGA is a synthetic copolymer composed of glycolic acid and lactic acid monomers. Lactic acid is a 2-hydroxypropanoic acid or methyl-substituted glycolic acid that can be produced in 2 forms i.e. D and L through fermentation of corn and other different agricultural sources [21]. Glycolic acid is 2-hydroxyethanoic acid that is produced either through a biochemical enzymatic reaction or by chemical synthesis using sodium hydroxide and chloroacetic acid. PLGA copolymer can be synthesized by various methods among which polycondensation reactions and ring opening polymerization are major ones [22]. Process parameters and reaction conditions greatly affect the physicochemical properties of the end product. That’s why to get polymer with desired characteristics; various mechanisms have been developed by doing slight modifications in process parameters of already available methods. These modified methods have helped to overcome the drawbacks associated with previous methods [23]. Different mechanisms for synthesis of PLGA along with their process parameters, drawbacks and advantages are summarized in table 1. Sequence of monomers in PLGA greatly influences the degradation rate, as random PLGA shows rapid degradation in comparison with sequenced PLGA. Application of sequenced PLGA in drug delivery, where the release kinetics of drugs are dramatically affected by polymeric degradation rate, is considered promising for controlled drug delivery [24].

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Properties of PLGA

Physicochemical properties of polymers are important for their characterization and determination of structure property relationships of polymers. Important physicochemical properties of biodegradable polymers include hydrophobicity, molecular weight, crystallinity, biodegradability, surface charge, composition of the co-polymer and glass transition temperature [31]. Amorphous PLGA copolymers have glass transition temperature in range of 45 to 55°C thus glassy in nature and exhibit fairly rigid chain structure. It has solubility in large variety of solvents like ethyl acetate, dichloromethane, tetrahydrofuran, hexafluoro-isopropanol, chloroform, benzyl alcohol and acetone. These copolymers degrade rapidly as compared to their counter homo polymers i.e. poly glycolic acid (PGA) or poly lactic acid (PLA). Various experiments have explained that PLGA gets softened in moistened environment even though it is hydrophobic in nature [32]. Molar ratio of the individual monomer components (lactide and glycolide) in the polymer chain directly influences many properties of PLGA such as degree of crystallinity, mechanical strength, swelling behavior, glass transition temperature and capability to hydrolyze. PGA does not contain methyl side chain and is highly crystalline in nature as compared to PLA. PLA is more hydrophobic due to methyl side chain in contrast to PGA, that’s why PLGA copolymers with high lactide content are less hydrophilic and degrade more slowly. It is well known that PLGA copolymer with a 50:50 molar ratio of the two monomers is hydrolyzed much faster in comparison with the one containing higher quantity of either of two monomers [33]. Particle elasticity like other physical parameters can also affect the drug delivery process. Elasticity can be modulated by addition of more polymeric layers and by increasing cross linking density. Harder 6

particles show increased cellular internalization and soft particles show longer circulation in blood. For example core-shell type PLGA-lipid spheres (PLGA core, lipid shell) with higher values of Young’s modulus were harder in nature showed improved internalization by HeLa tumor cells resulting in 2-fold increase in cellular death [34]. PLGA has either acid or ester as terminal group. Some products have both acid and ester end groups. Those having ester as terminal group offer resistance to hydrolysis. Various brands of PLGA including RESOMER®RG, DL-PLG and PDLG are available in different ratios of monomers i.e. 50:50, 65:35, 75:25, 85:15 having different physicochemical properties [35]. Ratio of monomers in PLGA can affect its amphipathic properties like lipophilic polymeric emulsification property. A study reported that PLGA with higher glycolic acid content has greatly improved the emulsifying properties of PLGA in protein loaded solid lipid nanoparticle preparation resulting in improved encapsulation efficiency and stability of emulsion [36]. In order to improve formulation properties of PLGA such as drug stability, degradation, release profiles and possibility of drug targeting, various structural modifications can be done. For example various PLGA copolymers including block and alternating polymers have been established with different hydrophilic moieties like poly ethylene glycol (PEG) or poly ethylene oxide (PEO) for aqueous solubility enhancement. Triblock copolymers thermosensitive in nature for protein delivery and triblock copolymers for enhancement of gene transfection efficiency of several cationic vector systems have also been developed. PLGA has also ability to attach different targeting moieties for example integrin-binding peptide was attached as contrast agent for cancer diagnosis. PLGA can be attached with groups containing positive charges for enhancement of cellular adhesion and uptake. To improve the hydrophilicity of polymer, PVA graft PLGA (PVAg-PLGA) was designed which showed the shift in degradation mechanism from bulk erosion to surface erosion. Polymers with increased anionic charge were obtained by linking sulfobutyl groups covalently to PVA backbone (SB-PVA-g-PLGA). Positively charged copolymers were obtained by linking various amino groups like dimethylaminopropyl amine (DMAPA), diethylaminopropyl amine (DEAPA) and diethylaminoethyl amine (DEAEA) to the PVA backbone [37]. Figure 1 illustrates how modulation of formulation properties of PLGA plays its role in enhancement of drug delivery. 4. Biosafety of PLGA Polymers are one of the four major categories of biomaterials. Biomaterials are very carefully selected for development of delivery systems considering the environmental conditions where it has to be targeted. The embedded material should not produce any untoward events such as inflammation, allergy and toxicity. Biocompatibility is a primary characteristic of a polymer indicating its worth to be used in biomedical field. Biocompatibility has been explored extensively with increasing interest to make assessment of properties for medical devices. Biocompatibility addresses many particulars of the biomaterials, including their physical, chemical and mechanical features, furthermore, mutagenic and allergenic effects, with objective to prevent any worth noticing unwanted effects on normal biological functions of cells [38]. Toxicity effects of PLGA NPs have been determined on wide variety of cell lines including Caco-2, Colo 205 and MDBK cells showing very little or no toxicity in-vitro [39, 40] and PLGA based nanostructures have also 7

been tested for their in-vivo toxicity on visceral organs ( liver, brain, spleen, intestine and kidney) after oral exposure of NP for less than 7 days. In recent study, the toxicity of PLGA and surfacemodified PLGA chitosan (PLGA/Chi) nanoparticles was assessed after oral administration for 7, 14 and 21 days to F344 rats. PLGA and PLGA modified chitosan showed similar bio distribution, illustrating minimal toxicity in intestine and liver, but no toxicity in kidney, brain and lung [41]. For immunotoxicity and genotoxicity testing of PLGA-PEO nanoparticles a human blood cell model was used. PLGA-PEO NPs were non-toxic in doses upto 3µg/cm2. Genotoxicity evaluation reported that there was no increase in micro nucleated binucleated cells (MNBNCs) and PLGAPEO treated cells were free of any oxidised DNA bases. Extensive experimentation with different particle size, composition and charge is needed to conclude the geno- and immunotoxicity of PLGA-PEO NPs [42]. 5. Polymer biodegradation PLGA is subjected to aqueous hydrolysis where polymer backbone having ester linkages is hydrolyzed randomly. Hydrolysis of each ester linkage results in one OH group and one COOH group. Reduction in the molecular weight due to scission of long polymer chains results in increased hydrophilic character; upon further reduction in molecular weight water-soluble polymer fragments are produced. These resulting fragments are further degraded to produce glycolic and lactic acids which are biologically inert to the growing cells and are eliminated from the body through common metabolic pathways [43]. 5.1 Factors affecting biodegradation of PLGA It is important to collectively apprehend the factors influencing degradation rate and drug release profile of polymeric drug composites. Proper accommodation of these factors in formulation of PLGA-based structures is also essential in order to achieve drug delivery systems with improved efficiency. For comprehensive understanding, these factors are classified into three major categories on the basis of material, processing, and physiological perspectives. Intrinsic physicochemical properties of polymer and drugs are included in material factors while processing parameters include the adjustable system design factors like size and shape of drug polymer composites, drug loading and monomer ratio. Factors affecting release environment like pH values, temperature, types of release medium and in-vitro/ in-vivo analysis are placed in group three as physiological factors [44]. Molecular weight (Mw) of copolymer greatly affects the both physical and chemical properties of PLGA. Typically molecular weight in range of 5 to150 k Da is used for controlled drug delivery systems. Generally, degradation rate and release of drug accelerate with decrease in Mw of PLGA [45]. PLGA-based structures have been extensively studied for delivery of a large variety of medicinal agents including macromolecules (like human growth factors, peptides, genes, vaccines, antigens, etc.) and comparatively smaller drugs (hydrophilic/hydrophobic). For hydrophobic drugs due to their low aqueous solubility, initial drug release is very low [46]. For example, it is reported that rate of diffusion for hydrophilic aspirin was 57.5 times higher than that of relatively hydrophobic haloperidol [47].

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Drug loading can also influence the drug release rate from polymer. Incorporation of higher initial drug amount results in increased rate of drug release [48]. Anyhow, at certain level of initial drug loading, effect of drug loading on degradation rate is diminished. However, there is not always a positive relation between initial drug loading and its release. As reported by a study that higher initial drug loading decelerates the diffusion rate of drug due to more compact internal structure that consequently obstructs the water absorption into the matrix [49, 50]. So, drug loading sometimes affect the degradation rate in positive or negative manner and sometimes it does not influence the release rate at all. Effect of each factor on polymer biodegradation and consequent drug release from polymer composites is summarized in figure 2. Each factor influences the rate of degradation either positively (may increase the rate) or negatively (decrease the rate) or in some conditions may not have any effect. 6. PLGA-based nanostructures and their formulation Different terminologies are being used to elucidate the nanostructures including nanocarriers, nanodisc, nanovehicles, nanoworm, nanosystem, nanotube, nanorod, drug polymer conjugates, liposomes, drug protein conjugates, dendrimers, polymer micelles and drug nanocrystals [51]. Nanostructures offer far-ranging advantages like reduced particle size improves the cellular penetration, increased drug entrapment efficiencies, and lesser minimum inhibitory concentrations [52]. From last decade researchers have synthesized PLGA nanoparticles using nano precipitation method [53, 54], emulsification-diffusion method [55], emulsification-evaporation method [56, 57] and solvent evaporation [58]. Each technique has its own pros and cons, but the fundamental rule to select the fabrication method depends upon physicochemical properties and possible interactions of drugs with polymers, organic solvents and surfactant, furthermore, the ultimate usage of established nano system. Methods that involve organic solvent diffusion to formulate PLGA nanoparticles limit the polymer concentration in order to keep the average size of 200 nm. Methods involving solvent evaporation come up with issues like time consumption and cost, but pose less sensitivity for polymer concentration changes. Emulsion evaporation method has been used to entrap both type of drugs i.e. hydrophilic (w/o/w emulsion) and hydrophobic (w/o emulsion). At higher concentrations of polymers, salting out method is preferable but owing to its exhausting purification process its use is limited. Concentration of polymer and surfactants, polymer molar mass, selection of solvents and phase ratios play a vital role in steering the particle’s size in all techniques employed for nanoparticle’s fabrication [59]. Different types of PLGA based nanostructures with their formulation techniques are summarized in table 2.

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7. Applications of PLGA in drug delivery

Delivery of drugs and various therapeutic agents is the basic approach of current medicine, following the idea that a disease is caused by deviant or diseased cells in healthy organs of body. For drugs to affect divergent organs or cells in an effective manner, these have to circumvent physiological barriers including circulation to cells, tissues and organs to successfully reach the target cells. Drug targeting to specific diseased organs through a drug delivery system (DDS) may improve the safety and efficacy of therapeutic agents, and prevent any drawbacks associated with therapeutic agents including toxicity, poor aqueous solubility, instability and less organ specificity. Furthermore, targeted delivery is an advantageous property for diagnostic reasons because it reduces unwanted interruption in signaling and improves selectivity and sensitivity. Implementation of nanotechnology in medicine has resulted in development of nanoparticlemediated drug delivery systems (nano-DDS), which has modified the kinetics of diagnostic and therapeutic agents. Currently, PLGA is used for development of FDA-approved biodegradable polymeric nano-DDS. PLGA polymers have ability to encapsulate a variety of hydrophilic and hydrophobic agents and are being investigated for troublesome diseases [81]. Various synthetic techniques are being explored to improve the targeting potential of PLGA nanoparticles. Molecular imprinting (MI) is the latest approach used to enhance the molecular recognition of PLGA NPs and has resulted in highly specific transport of these nanoparticles to the desired target organs [82]. 7.1 Role of PLGA in drug delivery to CVDs Cardiovascular diseases (CVDs) including several disorders of heart, blood vasculature and stroke, still remain the most common cause of mortality in USA in spite of remarkable clinical furtherance. World Health Organization (WHO) has estimated that CVDs will result in almost 25 million deaths of all global mortalities by 2030 [83, 84]. Multifarious nanostructures-based drugdelivery systems especially made up of biodegradable PLGA with multiple-functionalities exhibiting diversity in their sizes, shapes and surface functionalization with a wide variety of electrostatic charges and bio-molecular conjugations are being established for applications in CVDs [85, 86]. One of the main focuses of applications of nanotechnology in cardiovascular exploration has been the controlled imaging and drug delivery to atherosclerosis, myocardial infarction, restenosis and other cardiovascular conditions. Restenosis that is narrowing of a blood vessel which leads to restricted blood flow is an adverse effect of endovascular procedures. To prevent this restenosis various drugs are used including cytotoxics, inhibitors of smooth muscle cells ‘s growth (e.g. paclitaxel, etoposides, cytarbine, doxorubicin), immunomodulators (bisphosphonates, steroids, cyclosporine A), platelet-derived growth factors (PDGF) receptor inhibitors (e.g. tyrphostins), antibiotics (e.g. fumagillin) and gene therapy. To achieve controlled release profiles of these biomolecules and genetic materials, by providing them protection from unwanted enzymatic degradation, these are encapsulated in polymeric nanoparticles. Inhibition of restenosis in balloon injured carotid artery is achieved in rats by developing PLGA based nanoparticles encapsulating AGL 2043 and AG1295, selective blockers of PDGF receptor protein tyrosine kinase [87]. In blood circulatory system development of thrombus causes blockage of vasculature leading to life-threatening diseases including peripheral arterial thrombosis, myocardial infarction, 10

deep vein thrombosis, ischemic stroke and pulmonary embolism. Various plasminogen activators for the treatment of these disorders have been established. Microspheres composed of PLGA and PEG copolymers loaded with plasminogen activator demonstrated that microspheres can retain drug efficiently and release it at the desired site in concentration greater than 4 mg/ml. The improved thrombolysis has been depicted by pressure-driven permeation to the interior of clot. But, when there is no hydrodynamic pressure, pores of fibrin clots show resistance for the entry of carriers sizing 1 μm or greater into the clot interior. Therefore chitosan (CS) and CS-GRGD coated, PLGA NPs loaded with PA were fabricated and investigated in a blood clot-occluded model for their thrombolysis ability. PLGA/CS NPs demonstrated very short time for clot lysis and PLGA/CS-GRGD NPs illustrated the highest percentage for digested clots. These nanoparticles demonstrated improved permeation by showing long-term adherence and aggregation to the clot font and interior [88]. In myocardial ischemia (MI), depletion of myocardiocytes and fibrosis occurs, leading to cardiac dysfunction. Delivery of proangiogenic cytokines, including growth factors like vascular endothelial, hepatocyte or fibroblast growth factor 1 and 2 (VEGF), (HGF), (FGF-1 and -2), as protein or DNA recombinant, stimulates neovascularization of ischemic heart tissue and improves dysfunction. However, problems associated with delivery of these growth factors have led to the administration of synthetic molecules capable of stimulating endogenous proangiogenic cytokines‘s secretions. A slow-release formulation of prostacycline agonist ONO-1301 (SR-ONO) was developed by polymerization of ONO-1301 with PLGA. The present study showed that ONO1301 stimulated the endogenous production of HGF and significantly improved the recovery of perfusion in rat hind limb. Furthermore, epicardial injection of SR-ONO in swine chronic ischemia, encouraged collateral formation, promoted local wall motion of ventricles, and reduced the enlargement of left ventricle. PLGA can be absorbed gradually without damaging the tissue. Local delivery of SR-ONO has not induced any adverse effects that are associated with its oral delivery including diarrhea, hypotension and tachycardia [89]. Angiogenic therapy of myocardial ischemia with vascular endothelial growth factor (VEGF) is a favorable approach to overcome hypoxia and its sequel effects. PLGA particles loaded with VEGF have been proved a promising system for delivery of cytokines to rat myocardial ischemic model. This approach could be further explored for clinical studies [90]. The riveting proof for the role of oxidative stress in MI persuades the use of antioxidants. Coenzyme Q10 (CoQ10) owing to its role in mitochondrial electron transport chain appears to be a reliable candidate to treat MI but its poor biopharmaceutical characteristics needed to be addressed by developing promising delivery approaches. PLGA based nanoparticles were developed to encapsulate CoQ10 to overcome its poor pharmaceutical properties and administered to MI induced rats. Cardiac function was analyzed by determining ejection fraction before and after three months of therapy. Results showed significant betterment in the ejection fraction after three months [91]. In order to achieve better clinical outcomes, early reperfusion approach has been proved a standard strategy to reduce myocardial infarction size in MI patients. It is reported that reperfusion of coronary arteries results in paradoxical death of cardiomyocytes that is called myocardial ischemia-reperfusion (IR) injury. Inflammatory mediators specifically monocytes are important causative agents for this pathogenesis suggesting these as potential target for treatment of IR injury. PLGA nanoparticles were developed to incorporate irbesartan, an angiotensin receptor blocker (ARB) and were tested in mouse model 11

with IR injury. Single IV dose of PLGA nanoparticles showed better distribution to the monocytes and myocardium of the heart of the mouse. Efficient inhibition of the recruitment of monocytes in the IR heart and significantly reduced infarct size has proved the irbesartan nanoparticles a successful approach to treat myocardial IR injury [92]. Recent advancements in interventional cardiology engaging PCI and several other strategies of revascularization resulted in improvement of symptomatic CVDs. However, atherosclerotic disease is still a prime reason for death worldwide. Development of new therapeutics capable of intervening specific underlying molecular mechanisms responsible for disease pathogenesis is needed for improved patient’s prognosis. Establishment of different genetic models including atherosclerotic heart disease has led to important idea of gene therapy to fabricate new therapeutics for CVDs. Inflammation in the vascular wall involving monocytes is a distinctive feature for development of atherosclerosis. That’s why, monocyte chemo attractant protein-1 (MCP-1) and its receptor chemokine receptor 2 (CCR2) are suitable focus for gene therapy to prevent monocyte-regulated inflammation in atherosclerosis. 7ND a mutated MCP-1 lacking N-terminal 7 amino acids is capable of binding to CCR2 and blocking monocyte chemotaxis mediated by MCP-1. PLGA has been used to fabricate a suitable delivery system for genes. Gene therapy using 7ND plasmid encapsulated in PLGA nanoparticles has demonstrated atherosclerosis inhibition in mice having hypercholestremia. Ultimately, gene therapy using an avant-garde NP-regulated gene delivery system targeting MCP-1/CCR2 signals is a promising therapeutic approach to cure cardiovascular diseases [93]. 7.2 Applications of PLGA to treat inflammatory diseases In the clinical progression of advanced atherosclerotic lesions, chronic, non-resolving inflammation is a critical factor. Collagen IV (Col IV)–targeted PLGA nanoparticles encapsulating Ac2-26 (able to mimic pro-resolving actions of annexin A1) were tested for their therapeutic efficacy in chronic, advanced atherosclerosis after administration to mice. Col IV–Ac2-26 PLGA NPs have led to significant improvements in suppression of oxidative stress, advanced plaque properties and in reduction of plaque necrosis suggesting that targeted delivery of resolutionmediating peptide resulted in activation of receptors on myeloid cells for stabilization of advanced atherosclerotic lesions [94]. PLGA nanoparticles containing super paramagnetic iron oxide nanoparticles were also used to treat inflammation of joints indicating potential to avoid induction of inflammatory responses in joint diseases [95]. Pegylated PLGA nanoparticles have shown less exchange with mononuclear phagocyte system resulting in less induction of immune responses [96]. Inflammatory bowel disease (IBD) can be used as collective term for a group of chronic relapsing diseases of gastrointestinal (GI) including ulcerative colitis (UC) and crohn’s disease (CD). Cycles of remitting and relapsing mucosal inflammation are characteristic feature of UC and CD. Entire colon is commonly affected in both diseases requiring colon specific delivery of drugs to treat inflammatory bowel disease (IBD) [97]. Chitosan (CS)-modified PLGA nanospheres (NS) with a nuclear factor kappa B (NF-kB) decoy oligonucleotide (ODN) oral delivery system was developed to evaluate its use in an experimental model of ulcerative colitis (UC). Decoy ODN-loaded CS-PLGA NS resulted in improved stability of ODN against DNase I and gastric juice hence significantly enhanced cellular 12

uptake, efficient improvement in diarrhea induced by dextran sulfate sodium, shortening of colon length, bloody feces, and myeloperoxidase activity indicating that CS-PLGA NS can be an effective strategy for colon-specific oral decoy ODN delivery in UC [98]. Adverse drug reactions associated with oral administration of anti-inflammatory and immunosuppressive drugs to treat IBD can be reduced by using targeted drug delivery systems of nano size range having ability to specifically accumulate in inflamed mucosal areas. Different polyethylene glycol (PEG)-surface functionalized PLGA micro- and nanoparticles have showed an increased translocation and deposition in inflamed mucosa and were qualified as an innovative strategy to treat IBD [99]. 7.3 Applications of PLGA to treat infections Revelation of different approaches for development of delivery systems targeting infected cells has gained a noteworthy attention because eradication of intracellular infections is quite difficult. 18-β-glycyrrhetinic acid (GLA) loaded PLGA nanoparticles were developed with improved physicochemical properties. Results have revealed that the nanoparticles were found to be more efficacious against P. aeuroginosa, S. epidermidis and S. aureus than free GLA. Improved GLA’s antibacterial activity as compared to pure form might be attributed to higher penetration of GLA in infected cells [100]. Leishmaniasis a protozoan infectious disease presents a wide variety of clinical indications ranging from mild cutaneous ulcers to appalling visceral leishmaniasis. PLGA-PEG nanoparticles were fabricated to encapsulate amphotericin B to enhance its solubility and for targeting of macrophages of tissues infected by visceral leishmaniasis. PLGA-PEG encapsulating amphotericin B nanoparticles have showed more in-vitro and in-vivo efficacy than free amphotericin B [101]. HIV-1 pandemic is still an unrivalled public health issue, with new incidences of 2.1 million in 2013 and an approximately 35 million people are infected already. Most of the new infections reported are due to Vaginal HIV transmission. Currently, different antiretroviral drugs are administered prophylactically to prevent transmission of HIV. PLGA has been used to develop two innovative nanoformulations of rilpivirine for HIV prevention. PLGA based thermosensitive gel of rilpivirine, provided protection to mice from a high-dose HIV-1 challenge. A different PLGA based nanosuspension of rilpivirine, upon intramuscularly administration has protected mice from a vaginal high-dose of HIV-1 challenge. Results demonstrated that both topical and systemic formulations of rilpivirine has offered noteworthy coitus-dependent or independent immunity from HIV infection [102]. A DNA vaccine is effective to protect infection by infectious haematopoietic necrosis virus (IHNV) in rainbow trout but its administration through intramuscular route poses practical problems. PLGA based nanoparticles to encapsulate DNA vaccines were developed to administer through oral route. A virus challenge determination demonstrated an increase survival in fish after 6 weeks of post-vaccination with high dose oral vaccine [103]. Brucellosis, caused by brucella abortus is an infectious pandemic disease affecting over 500,000 new human cases annually. Absence of any reported human vaccine requires an effective vaccine. PLGA is used to develop vaccine delivery system carrying rL7/L12 protein encapsulated in it. Determination of the formulation’s immunogenicity in mice demonstrated increase in antibody titre up to 2.2 × 105 after the booster dose. Both humoral and cellular immunity has been achieved through this system [104]. Biofilm-forming bacteria cause life threatening infections in hospitalized patients including 13

cystic fibrosis and chronic obstructive pulmonary disease (COPD). DNase I functionalized, ciprofloxacin-loaded PLGA nanoparticle, was developed and assessment of antibiofilm activity was made against biofilms of Pseudomonas aeruginosa. These carriers have created fundamental change in treatment of biofilm. These nanocarriers are capable of disassembling the biofilm by degrading the stabilizer of biofilm matrix. Furthermore , these have shown 95% reduction in biofilm establishment and eradicated more than 99.8% biofilms [105]. PLGA has been used widely for fabrication of vaccine delivery systems for various infections including tetanus and diphtheria. The controlled release of Tetanus Toxoid (TT) from PLGA microspheres has extensively been investigated. Release of vaccine from PLGA depends upon its degradation rate. Furthermore, release profile has been prolonged from 21 days to 52 days with increasing ratio of lactic acid from 50:50 to 75:25 and with increasing molecular weight of PLGA. A comparison between TT-PLGA microsphere and conventional aluminum adsorbed TT has demonstrated that a single dose of TT-PLGA carriers presented almost similar immunity as provided by conventionally administered multiple doses of aluminum adsorbed TT. Corynebacterium diphtheriae causes diphtheria that is manifested by the presence of pseudomembranes in the upper respiratory tract. PLGA in different molecular weights and PLA: PGA ratio is widely used to successfully fabricate vaccine delivery system against diphtheria [106]. Dengue, a most common mosquito borne disease is caused by dengue virus having 4 distinct serotypes DEN-1–DEN-4. Currently there is no vaccine available for dengue. The non-structural protein NS-1of DEN-2 was incorporated in PLGA/PEG carriers. Significant immunity responses were demonstrated by these carriers in mice indicating an improved survival rate [107]. 7.4

Applications of PLGA in drug delivery to CNS

Neurodegenerative diseases are wide spreading and aging of the population has resulted in increased prevalence. More than 35 million people are suffering from Alzheimer’s disease (AD) worldwide and it is expected to be doubled by the year 2050 [108]. Presence of the blood–brain barrier (BBB) hinders the drug’s penetration to the central nervous systems resulting in lack of efficacy of current treatments requiring development of new drug delivery systems. Polymeric nanoparticles specifically, of PLGA have been proved advantageous for this purpose. [109, 110]. Loperamide-loaded PLGA nanoparticles were prepared by using a low-energy method and evidenced that these can cross the BBB efficiently, upon functionalization of their surface with a monoclonal antibody for active targeting against the transferrin receptor [111]. Parkinson’s disease is also a neurodegenerative disorder. Poly (lactic-co-glycolic) acid (PLGA) nanoparticles were developed for effective delivery of nicotine to brain in order to achieve neuro- protection effect against parkinsonism induced by Reactive Oxygen Species ROS [112]. PLGA nanoparticles loaded with lorazepam were successfully fabricated by using nanoprecipitation method. In vitro drug release profiles were found almost similar to ex-vivo permeation results determined in nasal mucosa of sheep. Vero cell line was used to evaluate the safety of NPs through in-vitro cell viability test. Scintigraphy imaging was performed by radiolabelling of Lzp-PLGA-NPs with Technitium-99m to develop nose-to-brain bio-distribution pathway in rats [113]. In spite of extensive research to explore treatment for cerebral vasospasm, the outcome of patients of Subarachnoid Hemorrhage (SAH) remained catastrophic. The inflammation following 14

SAH is a critical underlying pathway for the establishment and preservation of early brain injury (EBI). PLGA is used to encapsulate curcumin to improve its solubility related concentration in order to achieve a powerful anti-neuroinflammatory response in the affront CNS. Delivery of nanocurcumin has reduced leukocyte chemotaxis and successive inflammation in SAH rodent model suggesting that nanoparticle of herb could be clinically useful to reduce apoptosis induced by SAH [114]. Resveratrol a polyphenolic compound having antiaging, cardioprotective, anticancer characteristics and protective effects against PD is widely present in grapes, blueberry, mulberry, cranberry and peanut sprouts. Encapsulation of it in PLGA nanoparticles protects conversion of trans from into cis non active form resulting in increased bioavailability for prolonged anti-parkison effect in a rat model for up to 4 days [115]. Alzheimer’s disease (AD) is one of the most prevalent neurodegenerative diseases that cause dementia. Deposition of βamyloid (Aβ) is the prominent feature of this disorder which results in development of extracellular aggregates. Curcumin shows anti-amyloidogenic effect, by preventing the development of new Aβ aggregates and dispersing the existing ones. Curcumin cannot cross BBB efficiently resulting in lesser uptake by brain. Therefore, PLGA NPs were developed to encapsulate curcumin to improve its ability to cross BBB. In-vitro studies in hippocampal cell cultures have not shown any toxicity. Moreover, significant reduction in Aβ aggregation as a result of curcumin loaded PLGA NPs has suggested that these NPs are promising carriers for efficient therapy of AD [116]. PLGA nanoparticles fabricated to deliver siRNAs and miRNAs are used extensively in Alzheimer’s disease in animal models. Foremost, several chemicals, proteins and nucleic acids can be incorporated in to the PLGA matrix to improve the properties of nanoparticles. The emerging nanotechnology based delivery systems have potential to be used for miRNA therapy in central nervous system’s diseases [117]. Yu et al. fabricated PEG-PLGA polymerosomes and decorated them with lactoferrin an endogenous ligand for lac receptors to improve its properties for efficient delivery to brain [118]. Employing the same approach of LacR, Yu et al. developed PEG-PLGA polymersomes, decorated with lactoferrin, and additionally containing a neuroprotective peptide S14G-humanin resulting in protection of Aβ treated rats from memory impairment in a dose-dependent manner [119]. Encapsulation of estradiol in PLGA NPs coated with P-80 have resulted in significantly increased concentrations in brain after 24h (i.e. 1.969 ± 0.197 ng/g of tissue) as compared to uncoated NP (i.e. 1.105 ± 0.136 ng/g of tissue) in a Alzheimer’s Disease rat model [120]. PLGA NPs decorated with an anti-Aβ Antibodies (IgG) and surface functionalization with chitosan has shown increased transcytosis across the BBB in cell monolayer [121]. Zhang et al. developed PEG-PLGA NPs encapsulating drug (fibroblast growth factors) for AD therapy with attached Solanum tuberosum lectin as targeting moiety which specificaly binds with N-acetylglucosamine expressed on the epithelial membranes of nasal cavity. Upon intranasal administration the brain concentration of growth factor was increased up to 1.79-folds resulting in improved memory of rats suffering AD [122]. Another study has proved that PLGA nanoparticles can be employed to design an efficacious and safest vaccine for Alzheimer’s disease. In this study, PLGA nanoparticles were designed to encapsulate Aβ1–15 amino acid containing peptide for immunization of balb/c mice by subcut or intranasal route. Mice has displayed elicited Abs titers against full Aβ [123].

15

Multiple sclerosis (MS) is a disease of the central nervous system in which demyelination occurs in progressive manner. Myelin sheath provides insulation, metabolic support to axons and supports the conduction of electrical signals along the axons. Demyelination results in loss of electrical conduction and irreversible neurodegeneration that requires an effective treatment approach. A promyelination factor, leukaemia inhibitory factor (LIF), is a cytokine that contributes in self-immunological tolerance. PLGA nanoparticles (surface functionalized with Abs against NG2 expressing on OPCs) are used to deliver LIF to promote the differentiation of precursor cells into mature oligodendrocytes capable of repairing myelin. Myelin repair was seen at both levels, improved myelin thickness per axon and greater number of myelinated axons suggesting that it is a potential approach to treat MS [124]. By using PLGA, GAL-loaded nanoparticles have been designed with higher encapsulation efficiency, controlled drug release profile, non-toxic effect and preserving pharmacological activity of GAL, thus prolonging its therapeutic effects. These GALloaded nanoparticles with enhanced features have become a promising drug delivery system for neurodegenerative diseases [125]. PLGA has also been used to fabricate nanopatterned scaffolds to enhance the stem cell’s therapeutic efficacy to cure brain injury and neurodegenerative diseases. Biophysical cues offered by nanotopographical features are being used to stimulate neurite extension and regulate differentiation of neural stem cell (NSC). DOPA (dihydroxyLphenylalanine)-coated biodegradable PLGA polymeric substrates were fabricated having nanoscale topographic features for enhanced differentiation of human neural stem cells (hNSC) and directed neurite outgrowth. Incorporation of nerve growth factor has resulted in further enhancement of differentiation of hNSCs, illustrating a combined effect of biochemical and physical signals on stem cell differentiation [126]. 7.5 Cancer therapy with PLGA based nanostructures 7.5.1. PLGA based nanostructures for anticancer drug delivery For last few decades increasing prevalence of cancer has attracted the scientists to develop novel nano carrier systems for targeted delivery of anti-cancers. Anticancer drugs might be loaded in these nanocarriers or linked to their surfaces to reduce toxicities to sensitive normal cells. A controlled and sustained release of drugs over a period of days or even weeks at target sites is possible [127]. Recently, polymeric nanoparticles of poly (D, L-lactide-coglycolide) (PLGA), has been widely explored for targeted therapeutics in cancer nanomedicine. Iron oxide nanoparticles encapsulated in PLGA-PEG copolymer were loaded with doxorubicin suggesting that these drug– polymer magnetic composite nanoparticles might be employed as controlled delivery systems for drugs. For this reason, these nanoparticles have become a potent chemo preventive and chemotherapeutic system for patients of lung cancer and could be suitable candidates for development of drug delivery system [128]. Mannose-functionalized PLGA based NPs were developed for loading of melanomaassociated antigens and attachment of Toll-like receptor (TLR) ligands (Th1immunopotentiators) and targeted to mannose receptors on antigen-presenting cells. This nanoparticulate system elicited the highest delay in tumor growth in murine B16F10 melanoma tumors in prophylatic and therapeutic conditions demonstrating that the multifunctional NPs have high cancer immunotherapeutic potential [129]. Garcinol (GAR) has recently been investigated for its potential 16

for antiproliferative activity in a wide variety of human cancerous cell lines. Although it’s biological activity is very promising, but its poor aqueous solubility was the main hindrance in its clinical application. To overcome this issue GAR was encapsulated in PLGA nanoparticles fabricated by nano precipitation technique using vitamin E TPGS as emulsifier. GAR-PLGA-NP showed increased cytotoxicity in various cancer cell lines including HepG2, B16F10 and KB. Scintigraphic imaging of radiolabeled GAR-PLGA-NPs showed increased cellular uptake in melanoma bearing mice. Biological evaluation of these NPs has confirmed their effectiveness in cancer treatment [130]. To improve antigen-specific anti-tumor immunity, inhibition of SOCS1 (suppressor of cytokine signaling 1) gene expression is necessary because it negatively regulates the APC-based immune response. PLGA NPs capable of simultaneous delivery of siRNA and tumor antigen for immunosuppressive SOCS1 genes were developed. PLGA (OVA/SOCS1 siRNA) NPs showed efficient uptake by BMDCs (bone-marrow-derived dendritic cells) and knockdown of SOCS1 in BMDCs by these NP resulted in greater manifestation of proinflammatory cytokine inclusive of TNF-α and interleukins (IL-6, IL-12 and IL-2), suggesting that this system can be an effective approach to achieve better immunotherapeutic effects of BMDCbased cancer therapy [131]. Multicellular spheroids (three-dimensional culture cells) distinctly imitate in-vivo tumorlike conditions of physiologic environment and can be used for development of anticancer chemotherapeutics. Docetaxel-loaded PEG modified PLGA nanoparticles combined with antiHER2 antibodies (scFv–Doc–PLGA–PEG) were tested for cytotoxic effects on both HER2overexpressing and HER2-underexpressing models. Targeted nanoparticles demonstrated enhanced necrosis of tumor spheroids indicating that scFv–Doc–PLGA–PEG nanoparticles can be used for targeting of cancers over-expressing HER2 [132]. Triple-negative breast cancer has great risk for distant metastasis and early recurrence. Absence of targetable receptors makes its treatment difficult in selective manner. To overcome this issue self-assembled porphyrin based micelles were established from a hybrid polymer of PEG, PLGA and porphyrin. These PLGA based bilayer micelles were loaded with two anticancer drugs for synergistic activity and showed improved accumulation in tumor. Upon near-infrared laser irradiation, porphyrins, released sufficient heat for photothermal therapy. Combined localized photothermal effect along with synergistic chemotherapy resulted in increased tumor regression with reduced systemic side effects [133]. Methotrexate (MTX) a widely used anticancer drug poses many challenges including poor bioavailability and dose-dependent side effects. Polymeric micelles of glycine-PLGA were developed by linking glycine to PLGA. This novel formulation exhibited pH-dependent release of MTX in cancer cells with 4 fold increases in bioavailability and 100% cytotoxicity. This novel nano DDS has been proved a better carrier for delivery of chemotherapeutics with great promises of improved pharmacokinetic profile and enhancement of efficacy [134]. 7.5.2 PLGA based gene delivery to treat cancer PLGA NPs have potential for efficient gene delivery in addition to drug delivery. These NPs have several advantages for gene delivery as compared to other carrier systems. Several challenges associated with these NPs for gene delivery can be addressed through modification of these nano carriers by using different strategies [135]. A novel strategy to circumvent antitumor 17

drug resistance is the use of siRNA-mediated RNA interference to inhibit the manifestation of efflux transporters. Surface functionalized with biotin, siRNA targeted to P-gp, paclitaxel loaded PLGA nanoparticles were investigated for the efficiency of simultaneous and targeted anticancer drug delivery, to circumvent tumor drug resistance. Dual agent nanoparticles showed significantly higher cytotoxicity in-vitro and in-vivo inhibition of tumor growth than nanoparticles loaded with paclitaxel alone [136]. PLGA-b-PEG nanoparticles were also employed for co-loading of antisense miRNAs i.e. miR-21 and miR-10b to reduce in-vitro and in-vivo proliferation of breast cancer cells in culture and in tumor xenografts respectively suggesting that metastatic properties of tumors cells can efficiently be blocked by antagonizing multiple miRNA activities [137]. The two membrane-bound proteins, focal adhesion kinase (FAK) and excessively glycosylated CD44 which are two membrane associated proteins have a significant contribution in proliferation of cells, angiogenesis, and cell apoptosis and in cancer metastasis. PLGA nanoparticles carrying short hairpin RNA (shRNA) for FAK and CD44, delivered to ovarian cancer targets demonstrated that concurrent inhibition of both these genes retards cancer growth more effectively than knockdown of each gene separately [138]. Emanating cell-based therapies including gene therapy is facing a major challenge of rejection of transplanted engineered or donor cells and tissues by host immune system. To solve this issue an alternative strategy is the encapsulation of living cells in macro or microcapsules to attain immune-isolation of the cells, improving viability of cells in patient’s body rather suppressing the patient’s immune system. PLGA based capsules were developed for the protection of entrapped cells from being destroyed by both the mechanical stress and host’s immune system [139]. To achieve the potential effects of gene therapy, establishment of robust, safe, non-viral methods of delivery remains a major challenge. The miR-145 is dys-regulated in numerous cancers and further in-vitro investigations have demonstrated its pro-apoptotic and anti-proliferative roles. Upon local administration of PLGA/ polyethylenimine (PEI)-mediated miRNA delivery system, greater antitumor effects were attained in HCT-116 colon cancer cell lines and xenographt bearing mice, indicating PLGA/PEI/HA is a promising vehicle for replacement therapy of miRNA [140]. 7.6 Applications of PLGA in theranostics Theranostics is an emerging field which combines diagnostics and therapeutics into single multifunctional formulations. Advancements in nanoparticle systems capable of providing the necessary functionalities have driven the attention of researchers to this field. By employing these promising nanomedicines, the concept of individualized medicine can be realized by specifying treatment strategies to the individual [141]. These nanoparticles with dual functionality containing contrast agents for diagnostic purposes are very effective for intratumoral administration [142]. PLGA particles composed of gold nanoparticles, dye incorporated in the shell, and a core containing perfluorohexane (PFH) liquid were administered to rabbits bearing metastasized squamous carcinoma in their lymph nodes. Upon laser irradiation, liquid core was activated resulting in a rapid damage to surrounding cells and necrotic regions confirmed by histology and electron microscopy. This research has proved the ability of PLGA particles carrying PFC liquids to be used as in-vivo theranostic agents [143]. PLGA particles containing silica-coated gold nanoparticles (GNPs) and PFC liquid were designed and characterized through photoacoustic (PA) 18

technique. Confocal fluorescence imaging showed that PLGA particles were efficiently internalized by MDA-231 breast cancer lines. Upon vaporization of PLGA particles, bubbles were produced inside the cells resulted in their destruction demonstrating the potential of GNPs-loaded PLGA/PFC particles to be used in PA imaging and optically-activated drug delivery systems [144]. PLGA has also been investigated for use in Photothermal therapy (PTT) to treat various malignant diseases. PTT induced by near-infrared (NIR) has gained attraction owing to its negligible absorbance by healthy normal tissues and comparatively deep penetration in tissues. To enhance effectiveness of PTT, phase-shift and NIR photoabsorbing PLGA nanocapsules were designed for MRI/US double-modal imaging-directed PTT. These nanocapsules have potential to be employed not only for increasing the temperature of local tumor but as binary modal contrast agents as well for both US and MR imaging. Infrared lamp induced phase transition activity was referred as NIRDV (NIR radiation droplet vaporization) [145]. For a theranostic purpose, superparamagnetic iron oxide (SPIO)-paclitaxel (PTX)/loaded PLGA nanoparticles were developed. Their efficacy as T2 contrast agent was demonstrated by relaxometry studies and phantom MRI. Paclitaxel (PTX)-loaded nanoparticles have a cytotoxic activity while SPIO have not shown any toxic effects in CT-26 cells. Both PTX-loaded nanoparticles with co-encapulation of SPIO and without SPIO encapsulation suppressed the in-vivo regrowth of tumors. Multifunctional nanoparticles have given an avenue to future nanomedicine to consider simultaneous drug delivery, molecular imaging, and real-time monitoring of therapeutic response [146]. Radioiodinated PEGylated PLGA-indocyanine capsules were synthesized as a theranostic (multi-functional) agent, to target cervical, breast and ovarian cancers. The designed compound, with fluorescence capability (from Indocyanine), encapsulated radionuclidic tracer (131I) for tracing and paclitaxel an anticancer drug for targeting, has shown bioaffinity and promising potential for in-vivo imaging and therapy in ovarian, breast and cervical cancer cell lines [147]. Oleate-covered, iron oxide particles encapsulated into PLGA (PLGA-Fe-NPs) were ladened with PTX to trigger drug release by magnetic fluid hyperthermia MFH. Peculiar nuclear magnetic relaxation dispersion (NMRD) behaviors of developed formulations indicated their greater heating potentials in alternating magnetic fields. Fastened lipophilic Fe-NPs in PLGA has maintained the Neel relaxation as the prominent relaxation, resulting in preservation of their efficiency in the intracellular environment [148]. Management of inflammatory diseases propounds challenges due to inherent patient variability and complexity of inflammation process thereby requiring patientspecific therapeutic interventions. Theranostics could facilitate real-time therapeutic efficacy and assessment of safety and toxicity directing towards individualized treatment strategies. Macrophages being an important component of inflammatory diseases play various roles in disease resolution and exacerbation. PLGA based nanostructures have been explored to target and modulate the disease homing characteristics and intrinsic phagocytic nature of macrophages leading to inflammation resolution in several diseases [149]. 8. PLGA nanotechnology based products in clinical trials and market The significance of nanotechnology based PLGA drug delivery systems cannot be denied as their performance is well demonstrated in extensive in-vitro and in-vivo lab animal studies. In spite 19

of extensive lab investigations of these PLGA NPs, their use in clinical practice is still hindered by safety aspect. In order to get complete safety profile, it is essential to conduct clinical trials. Along with already marketed PLGA based microparticles and microspheres including Atridox® for periodontal treatment, Sandostatin® for acromegaly and Lupron depot® for prostate cancer [150], some nanoparticles based PLGA products are also under investigation in clinical trials. Docetaxel loaded PEG-PLGA-PMSA nanoparticles are currently under clinical trial phase I. These PLGA NPs were developed to treat metastatic or advanced cancer. It is a research product of Bind Biosciences, Inc. and named as BIND-014 [151-153]. Paclitaxel loaded PLGA-PEG-PLGA; thermosensitive system has been developed to treat esophageal cancer. This intratumoral OncoGel™ has been reached in clinical trials phase IIb for evaluation of its efficacy [154]. Phosphorex Inc. supplies PLGA nanoparticles with trade name Degradex® PLGA. These are plain PLGA nanoparticles that can be used for loading of any API. Phosphorex also provides fluorescent PLGA nanoparticles that can be used for imaging (http://degradex.com/about-degradexregproducts.html). A large number of novel PLGA NPs systems are still waiting for their clinical evaluation in order to reach market. 9. Conclusion and future prospective PLGA has been used successfully to develop nanostructures based drug delivery systems for various biomedical applications. Extensive in-vitro and in-vivo testing has proved the concept of potential advantage to use PLGA-based nanostructures in the treatment of various disorders. PLGA nanostructures offer a very suitable medium to carry out molecular level modifications such as the site specific imaging and targeting. In addition to targeted drug delivery to dreadful diseases like cancer and CVDs it is also being explored for its applications in vaccine delivery and tissue engineering. Therefore, in future, PLGA biodegradable polymer is going to be a perpetuate interest of researchers for applications not only in cancer therapy and theranostics but also in the field of biomedical engineering for tissue and nerve regeneration. The ingress of PLGA based DDS to clinical practice is only delayed but essential step. In future, it will not only be achievable but also worthwhile. References 1. 2. 3.

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28

Figure 1. Role of properties of PLGA in enhancement of drug delivery.

29

Figure 2. Factors affecting the degradation of PLGA and consequent drug release from polymer based drug delivery systems.

30

Table 1: Various routes for synthesis of PLGA

Ring opening polymerization Segmer assembly polymerization

Various routes for synthesis of PLGA

Polycondensation process

Starting material

Sub types Direct solution polycondensati on Azeotropic polycondensati on

Melt polycondensati on

Metal catalysts

Enzymatic ring opening

Process parameters > 120 ºC, Water removal condition Higher boiling point, Diphenyl ether

Without azeotropical solvents

Advantages / disadvantages Disadvantage: Mol. weight < 10 k Da Advantage: High mol. weight Disadvantage: Complex process control and purification, expensive

Advantage: High mol. weight, no environmental pollution 130-220 ºC Advantage: High mol. weight Tin (II) 2ethylhexanoate, Disadvantage: Contamination with tin (II) toxic metallic alkoxides, residues, or stannous random/atactic octoate PLGA (SnOct2) Advantage: Mild reaction conditions, environment Mild friendly temperature, Disadvantage: pH and Long reaction time, pressure larger quantities of Lipase enzymes required, random /atactic polymer 1,3 Advantage: diispropylcarbo Repeating sequence diimide (DIC), PLGA copolymer is 4(dimethylami obtained with no) pyridinium highly tunable psequence/ toluenesulfonat stereochemistry and e (DPTS) degradation kinetics

Ref.

[25]

[26]

[27]

[28]

[29]

[30]

31

Table 2: PLGA based nanostructures and their formulation techniques

32

Fabrication technique

PLA : PGA in PLGA/ Mol. weight

01

Chitosan/PLGA nanoplexes (160 nm)

Nanoprecipitation

75:25 66.000–107.000 Da

02

Nanobody conjugated PLGA nanoparticles (<200 nm)

water-in-oil-inwater (w/o/w) double emulsion/solvent evaporation

50:50 12000 Da

03

PLGA nanofiber scaffolds (400-500 nm dia)

Electrospinning techniques

50:50 48,000-Da

Solvent emulsionevaporation method

50:50 14, 500 Da

Moxifloxacin/ tuberculosis

Emulsionprecipitation method

75:25 5,000 Da

Paclitaxel and epigallocatechin gallate (EGCG)/ Breast cancer

Serial No.

Nanostructures

04

05

PLGA-PEG-chitosan nanoparticles (< 100 nm) PLGA casein core/shell nanoparticles (< 200 nm)

Drug used / Disease targeted

Result

High transfection efficiency, Improved in-vivo therapeutic efficacy against human multiple myeloma xenografts 7-fold decrease in IC50 of the formulation relative to Pentamidine/ African free drug, trypanosomiasis 10 fold reduction in therapeutic dose Remarkably enhanced cell viability and differentiation Human endometrial of stem cells into motor stem cell (hEnSC)/ neuron-like cells on PLGA Motor neuron diseases scaffolds as compared to tissue culture polystyrene (TCP) miR-34a (micro RNA)/ Multiple myeloma

Ref.

[60]

[61]

[62]

Sustained release, Reduced liver sequestration

[63]

Enhanced anti-NF-κB effects of EGCG resulted in improved Ptx therapy

[64]

33

06

PLGA nanobubbles (327 nm)

Double emulsion, solvent-diffusionevaporation

50:50 15000 Da

Doxorubicin and P-gp siRNA/ Breast Cancer

Increased cellular uptake and enhanced nuclear accumulation of DOX

[65]

50:50 15000 Da

Superparamagnetic iron oxide nanoparticles (SPION),manganesezinc sulfide (Mn:ZnS) quantum dots (QDs) and Busulfan (anticancer)

Enhanced drug delivery and improved imaging

[66]

07

PLGA polymeric vesicles (93 nm ± 20)

Emulsion evaporation method

08

Monocyte cell membrane-derived nanoghosts (<200 nm)

Nanoprecipitation and serial extrusion

NA

Doxorubicin/ Breast cancer

09

PLGA nanosuspensions (164–490 nm)

Solvent evaporation technique

75:25

Moxifloxacin/ corneal infection

Dendrimer-PLGA based immunenanocomposite (100-200 nm) PLGA–PEG–PLGA based nanogels (20- 250 nm)

Double emulsion solvent evaporation method

50:50 30-60 kDa

Cisplatin and siRNA/ Hepatocellular carcinoma

Co-assembly

NA

o/w emulsification and solvent evaporation technique

50:50 18,000 Da

10

11

12

HA Decorated PLGA nanoparticles ( 173.3 ± 2.2 nm)

Sufficient serum stability for 120 h, enhanced target specificity and greater cytotoxicity in metastatic cell lines (MCF 7) Improved permeation in goat cornea and enhanced antimicrobial activity against S. aureus,P.Aeruginosa

[67]

[68]

Synergistic anti-cancer effect

[69]

Taxol/ Cancer

Efficiently reduced tumor growth

[70]

Docetaxel and tanespimycin/ Cancer

Enhanced in-vitro and invivo synergistic antitumor activity

[71]

34

13

14

Lipid shell PLGA NP/liposomes (40 nm) Self-assembled PLGA/HA nanoparticles (< 200 nm)

15

Protein loaded PLGA nanoparticles (< 200 nm)

16

Triple-layered PLGA composite membrane

17

18

19

20

PLGA-PEG nanoparticles (70-300 nm) HNTs and CNTs doped composite PLGA nanofibers (< 1000 nm) EpCAM aptamer conjugated PEG– PLGA nanopolymersomes (< 120 nm) PLGA/polyethylenei mine nanoparticles based polyplexes

Microfluidic platform

_

_

Solvent-dialysis method

50:50 13, 600 Da

Docetaxel/ Breast cancer

50:50 24–38 kDa

Ovalbumin/ Vaccination

85:15

Lauric acid and nanoapatite/ Periodontitis

Focused ultrasound in an emulsion solvent diffusion method Solvent casting approach with thermally induced phase separation/ solvent leaching technique Double emulsion method

75:25

Curcumin/ Breast cancer

Electrospinning techniques

50:50 81,000 Da

Artificial tissue engineering

pH gradient method

75:25 10,000 Da

Doxorubicin/ Breast adenocarcinoma

w/o/w double emulsification/ solvent

65:35

pH1N1 DNA vaccine

Enhanced rigidity of NP leading to efficient cellular uptake

[72]

Enhanced anti-tumor activity with improved targeting of tumor

[73]

Scalable method for development of protein loaded nanoparticles

[74]

Efficient cell viability for human skin fibroblast

[75]

Enhanced cytotoxic effects on the breast cancer cell line

[76]

Improved mechanical strength of PLGA nanofibers

[77]

Controlled and targeted delivery of drug

Increased immune response as compared to naked DNA vaccine

[78]

[79]

35

21

coated on microneedles PLGA nanoparticles (110.0 ± 41.0 nm)

evaporation method Antisolvent diffusion method

75:25 10,000 Da

Estradiol/ Osteoporosis

Fast recovery of mineral density of cancellous bone

[80]

36