PLGA-based nanoparticles: A new paradigm in biomedical applications

Accepted Manuscript Title: PLGA-based nanoparticles: a new paradigm in biomedical applications Author: Shweta Sharma, Ankush Parmar, Shivpoojan Kori, Rajat Sandhir PII: DOI: Reference:

S0165-9936(15)30012-1 http://dx.doi.org/doi: 10.1016/j.trac.2015.06.014 TRAC 14581

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Trends in Analytical Chemistry

Please cite this article as: Shweta Sharma, Ankush Parmar, Shivpoojan Kori, Rajat Sandhir, PLGA-based nanoparticles: a new paradigm in biomedical applications, Trends in Analytical Chemistry (2015), http://dx.doi.org/doi: 10.1016/j.trac.2015.06.014. 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.

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PLGA-based Nanoparticles: A New Paradigm in Biomedical Applications

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Shweta Sharma*, Ankush Parmar, Shivpoojan Kori and Rajat Sandhir

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Institute of Forensic Science & Criminology

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Panjab University, Chandigarh, INDIA 160 014

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*Corresponding author. Tel.: +91-172-2534121(O) +91-9872688577 (M)

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E-mail: [email protected]

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Highlights

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

Structure-property relationship of PLGA on the concept of developing nanoparticles

9



Surface modification for providing functional sites to improve surface properties

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

Nanotechnology based applications in the field of therapeutic medicine

11



Research in nano-probes for thergonastic and its potential future

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Abstract

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Three decades back polymers were first introduced as bioresorbable surgical devices. Since then polymer based

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nanoparticles have intrigued many research groups to be extensively used in a variety of fields. Nanocarrier formulated

15

with the US FDA and EMA approved biocompatible and biodegradable polymers are being explored for the controlled

16

delivery of various therapeutic agents. Amidst the various polymers synthesized for formulating polymeric

17

nanoparticles PLGA has enticed considerable attention. PLGA possess many alluring properties such as controlled and

18

sustained release properties, low cytotoxicity, long standing track records in biomedical applications, biocompatibility

19

with tissues and cells, prolonged residence time, and targeted delivery. The prime objective of this review is to

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comprehensively address the issues related to PLGA based nanoparticles with special reference to methods of

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preparation, characterization techniques, surface modification, mechanism of drug release and the pitfalls. The review

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also critically addresses the developmental aspects of PLGA based nanocarriers in terms of targeted drug delivery, and

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exploring their efficacy in vitro and in vivo.

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Keywords: Biodegradable; Poly (lactic-co-glycolic acid); Nanoparticles; Targeting; Sustained release.

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INDEX

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1. Introduction

28

2. PLGA

29

3. Methods for preparation of PLGA nanoparticles

30 31

I.

Emulsification solvent evaporation method (a)

Single emulsion method

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(b) Double emulsion method

33

II.

34

III. Emulsification solvent diffusion method

35

IV. Emulsification reverse salting out method

36

V.

37

VI. Dialysis

38

VII. Spray drying

39

VIII. Supercritical fluid technology

Phase separation (Coacervation)

Nanoprecipitation method (Solvent displacement)

40

(a)

41

(b) Rapid expansion of supercritical solutions into liquid solvents

Rapid expansion of supercritical solutions

42

4. Characterization techniques for nanoparticles

43

5. Surface modification of PLGA nanoparticles

44 45

(a) Polyethylene glycol PEGylation strategies

46

(i)

47

(ii) Activated conjugation

48

(iii) Ring opening polymerization

Direct conjugation

49

(b) Polysorbate

50

(c) Vitamin E TPGS

51

6. Mechanism of drug release from PLGA based drug delivery system

52

7. Physiochemical changes occurring in PLGA based drug delivery system

53

8. Drug release behavior

54

9. Factors affecting degradation

55

(a) Polymer composition and molecular weight

56

(b) Drug type

57

(c) Size and shape of the matrix

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(d) pH

59

(e) Drug load

60

10. PLGA mediated drug delivery for cancer treatment

61

11. Targeting strategies for efficient drug delivery

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(a) Passive targeting

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(b) Active targeting

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(i)

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(ii) Targeting of tumor endothelium

Targeting of cancer cells

66

12. Ligand anchored PLGA nanoparticles for cancer therapy

67

13. PLGA nanoparticles as thriving mediator

68

(a) Gene delivery For cancer treatment

69

(b) Diagnosis and imaging of cancer

70

(c) Therganostics of cancer

71

14. Pitfalls encountered with PLGA nanoparticle based drug delivery system

72

15. Conclusion

73

16. References

74 75

List of abbreviations DCC

N,N-Dicyclohexyl carbodiimide

DDS

Drug delivery system

DNA

Deoxyribose nucleic acid

EDC

1-ethyl-3-(3-dimethylaminopropyl carbodiimide)

EGFR

Epidermal growth factor receptor

EMA

European medical agency

EPR

Enhanced permeability retention

FDA

Food and drug administration

FTIR

Fourier transform infra red

GALT

Gut associated lymphoid tissue

MDR

Multi drug resistance

MMP’s

Matrix metalloproteinase’s

MPS

Mononuclear phagocyte system

MRI

Magnetic resonance imaging

NHS

N-hydroxy succinimide

NMR

Nuclear magnetic resonance

O/W

Oil-in-water

PEG

Poly ethylene glycol

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PGA

Poly glycolic acid

P-gP

P-glycoprotein

PLA

Poly lactic acid

PLGA

Poly (lactic-co-glycolic acid)

PNP

Polymeric nanoparticle

RES

Reticuloendothelial system

RNA

Ribose nucleic acid

SiRNA

Short interfering ribose nucleic acid

99mTC

Technetium 99m

Tg

Glass transition temperature

TPGS

d-α tocopheryl polyethylene glycol succinate

VCAM

Vascular cell adhesion molecule

VEGFR

Vascular endothelial growth factor receptors

W/O/W

Water-in-oil-water

XPS

X ray photoelectron spectroscopy

76 77

1. Introduction

78

In recent years nanoparticles have become extremely enticing for their application in the field of biomedical

79

sciences. Depending upon the nature of the polymer used in the formulation, these particles may be categorized into

80

two categories i.e. natural or synthetic. Delivery of various substances like vaccines, macromolecules as well as

81

hydrophobic drug to cells and various peculiar organs like brain, liver, lungs etc. can be achieved via these

82

nanoparticles, thus making them a multifaceted platform for targeted delivery [1]. Whilst, in order to be used as a

83

vector for drug delivery system a nanoparticle must possess some vital properties like biocompatibility, drug

84

compatibility and proper biodegradation kinetics. A Site specific action of the drug at a therapeutically optimal rate

85

and dose regimen can be attained by restraining the parameters like particle size, surface properties and release rate

86

during the synthesis and designing phase of the nanoparticles. Site specific delivery, cancer therapy, clinical

87

bio-analytical diagnostics, tissue engineered scaffolds and devices are some of the fields where polymer based

88

nanoparticles have shown their utilization [2]. For the synthesis of nanoparticles a variety of polymers have been used

89

but the copolymer PLGA has been widely used in this context. PLGA is a US FDA and EMA approved biocompatible,

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biodegradable and safely administrable polymer [3]. Langer and Folkman [4] were the first to evince the controlled

91

release of macromolecules via polymers. It wouldn't be unjust to say that their contribution in this field has proven to

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be a breakthrough and has opened up new realms in the field of novel drug delivery system. This innovation has led to

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the evolution of anti angiogenic drug delivery systems for the treatment of dreadful disease like cancer. There have

94

been reports where, PLGA nanoparticles have proven to be magnificent vector for the transmission of bio-molecules

95

such as RNA, DNA, peptides, vitamins, proteins and drugs both in vivo and in vitro. Stevanovic and Uskokovic have

96

highlighted the efficient delivery of vitamins using PLGA based nano as well as microparticles [5]. Choi et al., in their

97

review article, concisely elaborated PLGA aided tumour targeting[6]. In another report, Locatelli and Franchini

98

explained the synthesis and applications of PLGA-b-PEG polymeric nanoparticles [7]. A comprehensive compilation

99

has been given by Joo et al., where various features of PLGA including surface modification, targeting aspect and the

100

intrinsic capacity as cancer drug carrier has been taken up [8]. The Present review initially encompasses the detailed

101

aspects of PLGA and its associated potentials in terms of structure-property relationship. The advancement in the

102

efficacy of PLGA nanoparticles has been depicted through ligand anchoring, their application as mediator for gene

103

delivery, in imaging of cancer and in the field of therganostics.

104

2. PLGA

105

Polyester PLGA is a copolymer of PLA and PGA. With respect to design and performance PLGA is the best defined

106

biomaterial available for drug delivery. PLGA here stand for poly D, L-lactic-co-glycolic acid where D- and L-lactic

107

acid forms are present in a fixed ratio [9]. The urgency of an efficient and better drug delivery system has lead to the

108

development of assorted block copolymers of polyesters with PEG. PLGA/PEG block copolymers are available in two

109

varieties i.e. Diblock (PLGA-PEG) or Triblock with both ABA (PLGA-PEG-PLGA) and BAB (PEG-PLGA-PEG).

110

Properties of PLGA

111

Synthesis of PLGA is achieved via random ring opening copolymerization of two different monomers, glycolic acid

112

and lactic acid in presence of tin (II) 2-ethylhexanote, tin (II) alkoxides or aluminium isoproxide as catalyst. Glycolic

113

and lactic acid units are consecutively linked together via ester linkages during polymerization, resulting in the

114

formation of PLGA.

115

Different forms of PLGA can be obtained by using the varied ratio of lactide to glycolide during polymerization

116

reaction e.g. PLGA 50:50 (refers to a copolymer which comprises of 50 % lactic acid and 50 % glycolic acid), PLGA

117

75:25, PLGA 80:20 etc. Depending upon the molecular weight and lactide to glycolide copolymer ratio used the

118

deterioration time of polymer may vary from several months to several years[1, 10]. Low molecular weight polymers

119

having higher glycolide content are more hydrophilic and amorphous, thus possess shorter deterioration time. This

120

may be explained on the basis of the fact that glycolic acid is more hydrophilic and thus tends to absorb a large amount

121

of water. While, the polymers having higher lactic acid content are more hydrophobic and absorbs less amount of

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water and degrades more gradually[11]. This phenomenon has been proven to be handy for a controlled and sustained

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drug release varying from weeks to months. The hydrolysis of PLGA yields two metabolic monomers i.e. lactic acid

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and glycolic acid. It is through the krebs cycle that these monomers are endogenously metabolized into the simpler

125

by-products like CO2 and H2O[12]. PLGA can be processed into almost any kind of configuration and is capable of

126

encapsulating a variety of molecules. PLGA polymers are miscible in a variety of volatile organic solvents viz.

127

tetrahydrofuran, acetone, dichloromethane, chloroform and ethyl acetate. Physical properties like molecular weight,

128

polydispersity index, Tg and degree of crystallinity have shown to effect the swelling behaviour, biodegradation rate

129

and mechanical strength of the polymer[13]. The type and molar ratio of the individual monomer components in the

130

polymer chain is the prime factor which governs the degree of crystallinity.

131

Makadia and Siegel have deciphered [9] that a decrease in the degree of crystallinity along with an increase in the

132

hydration rate (hydrolysis) is observed when copolymerization of a crystalline PGA with PLA occurs. PLGA

133

copolymers are generally glassy in nature and exhibit a fairly rigid chain structure. This may be attributed to the fact

134

that the Tg of PLGA is above its physiological temperature[14]. It has been demonstrated that dose and composition of

135

PLGA nanocarrier has a demarcating effect on blood clearance and uptake of these nanocarriers by the mononuclear

136

phagocytic system[15].

137

3. Methods of preparation of PLGA nanoparticles

138

Depending upon the process of preparation, the structural organization of the nanoparticle may vary. Preparation of

139

biodegradable PLGA nanoparticles is generally attained by dispersing the polymer. The nanoparticles are actually

140

formed in the initial step which is common for all the techniques and comprises of the preparation of an emulsification

141

system[8]. The different methods of preparation have been discussed ahead:

142

I. Emulsification-solvent evaporation method

143

(a) Single emulsion method

144

This is the most commonly employed method for the preparation of PLGA nanoparticles. O/W emulsification is

145

generally used when the encapsulant is hydrophobic or poorly soluble in water[16]. In order to prepare an organic

146

phase an appropriate amount of polymer is firstly dissolved in a volatile organic solvent such as dichloromethane,

147

chloroform or ethyl acetate. Thereafter, the drug or encapsulant is added to this solution, resulting in dispersion. This

148

dispersion containing polymer and drug is added into a continuously stirring aqueous solution containing surfactants

149

like poly vinyl alcohol, polysorbate 80, poloxamer 188 and vitamin E TPGS, leading to the generation of a stable

150

emulsion. The organic solvent is then allowed to evaporate either by magnetic stirring or by maintaining a reduced

151

pressure[9].

152

(b) Double emulsion method

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Double emulsion method is also known as W/O/W method. The encapsulation efficiency and particle size are

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predominantly affected by the type of solvent and stirring rate. An appropriate amount of drug is dissolved in an

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aqueous phase (prepared in deionized water) followed by simultaneous addition of drug solution into a vigorously

156

stirring organic phase (comprising of PLGA dissolved in volatile organic solvents such as ethyl acetate, chloroform,

157

dichloromethane etc). This results in formation of a water-in-oil primary emulsion. Further emulsification is achieved

158

by adding the primary emulsion into an aqueous solution followed by simultaneous stirring and thereafter allowing the

159

organic solvent to get evaporated[17]. Though, both these techniques are optimal for laboratory synthesis but a large

160

scale production using these techniques is hindered by the facts that both the techniques are applicable only to

161

liposoluble drugs and during homogenization high energy is required. However, it has been reported that alteration in

162

process parameters e.g. stirring speed and temperature helps in overcoming the shortcomings associated with these

163

techniques[8].

164

II. Phase separation (Coacervation)

165

This process mainly leads to the formation of micrometer sized polymeric nanoparticles via liquid-liquid phase

166

separation. In this method, two liquid phases comprising of coacervate phase and supernatant phase depleted in

167

polymer are formed. The coacervation process includes three major steps[9];

168

(i)

Phase separation of the coating polymer solution

169

(ii)

Adsorption of the coacervate around the drug particles and

170

(iii)

Quenching of the microspheres

171

Polymers and solvent are mixed in an appropriate ratio, followed by the addition of the drug (i.e. hydrophilic drug in

172

W/O/W emulsion whereas hydrophobic drug in O/W emulsion). A soft coacervate of drug encapsulated in a droplet is

173

extracted as a result of phase separation of polymer. The micro droplets are quenched by dipping the coacervate

174

quickly into an insoluble medium. However, the morphology and size of the microspheres can be controlled by

175

altering the following process parameters[18];

176

(a) Polymer concentration

177

(b) Quenching temperature

178

(c) Quenching time

179

(d) Solvent composition

180

(e) Stirring rate

181

III. Emulsification solvent diffusion method

182

This technique is the modification of salting out process. An initial thermodynamic equilibrium is attained by

183

mutually saturating the solvent and water at room temperature prior to use. Emulsification of organic phase containing

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the polymer and drug in aqueous surfactant solution is achieved via high speed homogenization. In order to attain a

185

phase transformation reaction and outward diffusion of the solvent from the internal phase, water is added under

186

regular stirring. Colloidal nanoparticles are formed as a result of nanoprecipitation. Solvent evaporation is then

187

facilitated either by evaporation or vacuum distillation[19]. This method offers numerous advantages like enhanced

188

encapsulation efficiency, high batch to batch reproducibility, ease of scale up, narrow size distribution and simplicity.

189

However, on the contrary, this technique is also accompanied by number of flaws such as leakage of water soluble

190

drugs and removal of high volume of water from the suspension.

191

IV. Emulsification reverse salting out method

192

The organic phase containing polymer and drug is firstly added to a water miscible solvent. An O/W emulsion is

193

formed by forcefully magnetically stirring the preformed organic solvent with an aqueous solution containing the

194

salting out agent (e.g. magnesium chloride, calcium chloride) and a colloidal stabilizer (e.g. polyvinyl pyrollidone). An

195

abrupt increase in the continuous phase of the emulsion is facilitated by further diluting the O/W with plenty of water.

196

The diffusion of volatile organic solvent into the aqueous phase starts as a result of which nanoparticles are

197

formed[20]. The residual solvent and salting out agents are removed by filtration, leaving the nanoparticles behind.

198

This technique offers advantage for encapsulation of heat sensitive agents such as proteins, DNA, RNA etc.

199

V. Nanoprecipitation method (Solvent displacement)

200

It is a one step process, generally used to entrap hydrophobic drugs in the polymer matrix. The organic phase is

201

formed by dissolving polymer and drug in a polar solvent (e.g. acetone, ethanol, methanol and acetonitrile). This

202

solution is added in a drop wise manner to an aqueous solution containing emulsifier or surfactant. The rapid diffusion

203

of solvent takes place resulting in the formation of nanoparticles[21-23].

204

VI. Dialysis

205

Dialysis is generally employed for the preparation of small sized nanoparticles having a narrow distribution. The

206

polymer is dissolved in a volatile organic solvent and placed inside a dialysis tube of appropriate pore size. Inside the

207

dialysis bag displacement of solvent takes place, along with a loss in the solubility hence ultimately leading to the

208

progressive aggregation of polymer and formation of homogenous suspension of nanoparticles[21, 24].

209

VII. Spray drying

210

Spray drying is an alternative to the conventional approaches of polymer nanoparticles formation. This technique

211

offers several advantages such as rapidity, convenience and implementing fewer processing parameters. In this

212

process, W/O dispersion is sprayed in a hot stream of air, leading to the formation of nanoparticles. However, the

213

adhesion of the nanoparticles to the inner walls of the spray dryer hinders in effective collection of formed

214

particles[25].

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VIII. Supercritical fluid technology

216

Supercritical fluid technology has been proven to be a more environmental friendly approach for the production of

217

PNP. It has the potential to produce PNPs with high purity and fewer traces of organic solvent[26]. Two principle

218

methods are generally employed for the production of nanoparticles[27];

219

(a) Rapid expansion of supercritical solutions: A solution is formed by dissolving the solute in a supercritical fluid,

220

which is accompanied with the rapid expansion of the solution into ambient air. The homogenous nucleation is

221

achieved by reducing the pressure rapidly. Due to the rapid reduction in the pressure a high degree of super saturation

222

is achieved, leading to expansion and ultimately formation of nanoparticles[27].

223

(b) Rapid expansion of supercritical solutions into liquid solvents: It is a modified form of rapid expansion of

224

supercritical solutions, where expansion of the supercritical solution takes place in a liquid solvent instead of ambient

225

air. Apparently the particle growth is suppressed in the expansion jet, by the action of the liquid solvent, leading to the

226

formation of nanoparticles[27, 28].

227

4. Characterization parameters for nanoparticles

228

Characterization becomes a prerequisite for understanding the properties of the nanoparticles. A number of

229

parameters are available from which a fair idea about the properties of the nanoparticles can be drawn. The first and

230

the foremost is the size, which helps in determining the efficacy of the nanoparticles, release profile and degradation

231

pattern. Dynamic light scattering, scanning electron microscopy, transmission electron microscopy and atomic force

232

microscopy are some of the techniques from which the parameters like size, distribution and morphology of the

233

nanoparticle can be ascertained. It has also been revealed that the molecular weight of the polymer have an adverse

234

effect on the particle size, encapsulation efficiency and degradation rate[23]. The chain length of the polymer

235

represents the molecular weight of the polymer and gives a basic indication about the chemical nature of the polymer

236

i.e. whether it is hydrophobic or hydrophilic. It is well versed that polymers having shorter chain length are

237

hydrophobic and have a faster degradation rate. However, the polymers having a longer chain length are generally

238

hydrophilic in nature and have a shorter degradation rate. The molecular weight of the polymer thus, plays a very

239

requisite role in deciding the release kinetics of the drug[29]. Size exclusion chromatography is one of the techniques

240

which are proven to be handy in determining the molecular weight of the polymer[30]. It has also been observed that

241

the in vitro and in vivo release characteristic of the drug is affected by the physical state of both the drug and polymer.

242

The muco-adhesion, nanoparticle constancy as well as intracellular trafficking are greatly dependent on the zeta

243

potential[31]. The biodistribution of the nanoparticles is greatly dependent upon the hydrophobic nature of the

244

nanoparticle. It has also been corroborated by various studies that the retention time of hydrophilic particles is more in

245

comparison to the hydrophobic particles[32]. Techniques like water contact angle measurement and hydrophobic

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interaction chromatography may be used for determining the hydrophobicity and hydrophilicity of the nanoparticles

247

respectively[33]. The surface chemistry can be analyzed using a variety of techniques such as XPS, FTIR spectroscopy

248

and NMR spectroscopys[23, 34].

249

5. Surface modification of PLGA nanoparticles

250

In order to act as a targeted drug delivery vehicle, it is desirable that nanoparticle must persist inside the systematic

251

circulation of the body. A prolonged circulation time will facilitate the nanoparticles to reach the target organ. On the

252

contrary, these particles are removed from the blood stream by RES. This phenomenon has proven to be one of the

253

foremost obstacles in the nanoparticle based drug delivery system[1, 35]. It may be attributed to the fact that these

254

nanoparticles bind with the opsonin proteins present in the blood serum when administered intravenously. The

255

opsonized particles further get attached to the macrophages, where they ultimately gets internalized by phagocytosis.

256

The final fate of such particles is their clearance from the body via renal system. Therefore, despite having the

257

favourable biocompatibility and biodegradability PLGA nanoparticles are often amenable to rapid clearance from

258

circulation by macrophages of the MPS immediately after their administration through intravenous route[36]. In order

259

to circumvent these hurdles, the bare nanoparticles are imposed to surface modification. Surface modification plays a

260

very compelling role in escaping the natural defence system of the body. The hydrophilic particles with size of about

261

100 nm or less have the greatest survival rate in escaping the phagocytic system[23]. The retention time for

262

hydrophilic nanoparticles is comparatively longer than hydrophobic nanoparticle. It has been attributed to the fact that

263

hydrophobic nanoparticles are preferably taken up by the reticuloendothelial system and are thus eliminated from the

264

body[8]. In order to formulate a hydrophilic core around PLGA nanoparticles, they are coated with surface

265

derivatizers. Some of the chemical species acting as good surface modifiers are:

266

(a) Polyethylene glycol

267

PEG is a non ionic, hydrophilic polymer which exhibits exceptional biocompatibility. Coating the nanoparticle

268

surface with poly ethylene glycol i.e. PEGylation is the most commonly employed technique for surface modification

269

of the nanoparticles. The process enables the nanoparticles to evade the mononuclear phagocytic system attack, hence

270

providing a concomitant increase in their plasma half life[37]. However, the mechanism behind this increase in the

271

plasma half life is still not well understood. It is assumed that stearic repulsion as well as Vander Waal forces created

272

by the hydrated barriers present on the nanoparticles surface prevents the nanoparticles from getting opsonized. The

273

high flexibility of the polymer chain allows the free rotation of the polymer units, creating a highly hydrophilic stealth

274

corona around the nanoparticles, which prevent the interaction of the nanoparticles with the macromolecules present in

275

the body. It has also been observed that PEG molecules having high molecular weight, high surface density and longer

276

chain length are absorbed at a comparatively lower rate, resulting in increase in the residence time of the

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nanoparticle[38]. Apart from this, PEGylation of PLGA nanoparticles has also been shown to enhance the drug

278

payload, solubility and kinetic stability thereby improving the targeting index, therapeutic index as well as the

279

accessibility of the nanoparticle towards the target site. With the help of PEGylation the aqueous solubility and

280

stability can be enhanced, intermolecular aggregation can be reduced and immunogenicity can be decreased. In a

281

study conducted by Danhier et al. it has been shown that the entrapment of paclitaxel within a PEGylated-PLGA based

282

nanoparticle altered its pharmacokinetics and biodistribution in such a way that the tumour specific localization of the

283

drug was significantly augmented, resulting in higher tumour growth inhibition efficiency as compared to the free

284

drug[39]. In another experiment by Gref et al, it has been shown that the PEGylated PLGA nanoparticles result in an

285

increase in the circulation time and decreased uptake by liver i.e. uptake of nanoparticles was reduced from 66%

286

within first 5 minutes of administration whereas, on the other hand it was found out to be less than 30% (within 2

287

hours) for non coated nanoparticles[40]. Although long-circulating nanoparticles formulated with PEGylated PLGA

288

possess many significant advantages, but this strategy is also not free from limitations. Capping of PLGA with PEG

289

not only prevents the interaction between the nanoparticles and the opsonin, but also it prevents the interaction

290

between nanoparticles and the cell surface[36]. Table 1 shows the effect of PEGylation on uptake of PLGA

291

nanoparticles in cancerous cells[40-42].

292

PEGylation Strategies

293

(i) Direct conjugation:

294

Betancourt et al. has reported three different methods for the covalent attachment of PEG to PLGA nanoparticles[43].

295

It is deciphered from their studies that by varying the reaction conditions the efficacy of conjugation as well as the

296

final copolymer composition could be controlled. A non significant incorporation of PEG occurred when PEG

297

molecule was directly conjugated to the carboxylic groups present on the surface of PLGA nanoparticles, because of

298

the inaccessibility of these groups. Moderate efficiency is observed, when the conjugation reactions are carried out in

299

solution. Direct conjugation of PLGA nanoparticles with PEG would also facilitate the encapsulation of a desired

300

agent into the nanoparticle as PEGylation would occur after the desired agent is encapsulated within the solid polymer

301

matrix core (Fig.1(a)). However, the technique is accompanied with certain limitations such as low yield because of

302

the purification steps. The nanoparticles have to be exposed to an aqueous environment during the conjugation process

303

in order to achieve maximum efficiency.

304

(ii) Activated conjugation:

305

It is a two step process where activation is succeeded by conjugation. A minimal hydrolysis of the active

306

intermediate occurs and the undesirable formation of PEG-PEG conjugates is avoided[43] . Fig.1(b) shows the

307

synthetic route utilized for conjugation of heterofunctional PEG to PLGA nanoparticles.

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308

(iii) Ring opening polymerization:

309

Among all the strategies used for the production of PEGylated PLGA nanoparticles ring opening polymerization is

310

the most commonly and widely employed technique. The reaction is initiated by protic agents such as hydroxyl group

311

of OH-PEG-COOH, leading to the formation of PLGA with hydroxyl end groups whereas the carboxyl end groups of

312

PEG remains free. Fig.1(c) shows ring opening polymerization of PEG to PLGA nanoparticles.

313

(b) Polysorbate

314

Polysorbate 20, 40, 60 and 80 are the most commonly used non ionic surfactant and emulsifier often used in foods

315

and cosmetics. Due to the surface coating of polymer nanoparticles with polysorbate, their ability to cross the Blood

316

Brain Barrier is enhanced. Because of the binding of the nanoparticles with the inner linings of the brain capillaries a

317

large change in the concentration gradient is observed along with an increase in the passive diffusion, resulting in

318

facilitated delivery of the drug to the brain.

319

(c) Vitamin E TPGS

320

Vitamin E TPGS is a synthetic water soluble form of Vitamin E. TPGS is a polyethylene glycol derivative of

321

α-tocopherol that enables water solubility. It has been most commonly used as an emulsifier, a solubilizer and as a

322

vehicle in drug delivery formulations. Vitamin E TPGS has been most commonly used as an emulsifier for enhanced

323

encapsulation efficiency, drug loading and enhanced release kinetics of the hydrophobic drug like paclitaxel,

324

doxorubicin and 5-Fluorouracil. The molecule has shown to improve the nanoparticle adhesion to the cells and

325

hemodynamic properties of the nanoparticles[33].

326

6. Mechanism of drug release from PLGA based drug delivery system (DDS)

327

The term "release mechanism" may be defined as the way in which the drug molecules are transported or released

328

and as an event of the process determining the release rate. There are only three possible mechanisms through which

329

the drug molecules can be released[44];

330

(i) Transport through water filled pores

331

(ii) Transport through the polymer

332

(iii) Dissolution of the encapsulating polymer

333

Large and highly hydrophilic molecules like protein or DNA are some of the encapsulated agents which are generally

334

transported through the polymer phase and are released by transport through water filled pores. Transport through

335

water filled pores is mainly achieved via a process known as diffusion, where the molecules are driven by chemical

336

potential gradient. Another phenomenon of transport is convection, where osmotic pressure is the driving force and

337

hence the name osmotic pumping. This pressure may generate influx of water into a non swelling system. The

338

phenomenon is most commonly confronted in case of cellulose acetate derived drug delivery systems[45]. PLGA

12 Page 12 of 27

339

possesses mobile polymer chains and has a tendency to absorb a large amount of water as consequence of which it

340

shows prominent swelling. Due to rearrangement of the polymer chain and swelling, the increase in the volume of

341

water is compensated. Sometimes encapsulated drug may be released as a result of dissolution of the polymer or

342

erosion. Pores are created and an increase in the rate of diffusion is observed. At initial stage, the release rate is

343

controlled by diffusion whereas in the final stage of the release period degradation or erosion plays a very vital role. It

344

may thus be concluded that the drug release may be controlled by more than one true mechanism at once[44].

345

7. Physiochemical changes occurring in PLGA based DDS

346

When the DDS is administered in vitro or immersed in water, the polymer quickly absorbs water. The water

347

molecules occupy some volume in the polymer matrix which is often regarded as pores and the process is said to be

348

pore forming process. Owing to the small size of the pores, a minimal amount of drug is transported during the initial

349

release phase, but time span, the water filled pores start to grow in size and in number resulting in creation of a porous

350

connected network. This network facilitates the drug release during the later phase. However, heterogeneous

351

degradation via an auto catalytic phenomenon takes place when PLGA matrix comes in contact with water. This

352

phenomenon is termed as hydrolysis. During hydrolysis, the ester bonds are broken, with a decrease in the molecular

353

weight of the polymer and generation of acids[46]. With an increase in the dimension of the drug delivery system it

354

has been observed that a faster degradation occurs at the centre of the PLGA matrix than at the surface[47].

355

Furthermore, loss of polymer takes place when the dissolved polymer degradation products diffuse into the release

356

medium. Due to its tendency to get hydrated rapidly PLGA polymer undergoes bulk erosion in spite of surface erosion.

357

Pores are created by the dissolution of polymer degradation and erosion process. The small pores so generated grow in

358

size by coalescing with the neighbouring pores ultimately leading to the formation of larger pores. PLGA Polymer has

359

the ability to rearrange them by mobilizing their polymeric chains and it is by the virtue of this phenomenon that larger

360

pores are formed. The effect of dissolved degradation products on drug delivery system is as follows;

361

(i) Owing to their acidic nature, they catalyze hydrolysis

362

(ii) An increase in the rate of water absorption along with a decrease in the transport resistance of the polymer is

363

observed due to plasticization of the polymer by dissolved degradation product

364

(iii) Osmolality inside the polymer matrix is increased thereby enhancing the force of water absorption

365

(iv) Due to the presence of many repeating monomeric units in a row crystallization takes place as a result of which the

366

absorption of water is inhibited

367

The effect of dissolved degradation products on the release of the polymer ceases with the onset of rapid erosion.

368

The transport resistance plays a very paradigm role in governing the release of the encapsulated moiety and polymer

369

degradation kinetics. Transport resistance is found to be affected by various processes such as pore formation, pore

13 Page 13 of 27

370

closure, drug dissolution, polymer–drug interaction and drug–drug interaction[44]. It has also been deciphered that an

371

increase in the density of the particle and reduction in the porosity (structural relaxation) might result in a decreased

372

burst release of drug molecule from the PLGA nanoparticles. The magnitude of the drug release in a particular

373

formulation is directly affected by the rate and extent of structural relaxation present in the formulated nanoparticle.

374

Structural relaxation in PLGA nanoparticles is dependent on various properties like molecular weight, fabrication

375

methods, drug polymer interactions, residual solvents and storage conditions. These factors all together contribute to

376

structural relaxation. They may also lead to the variability in burst release that impedes the development of products

377

using this type of drug delivery technology.

378

8. Drug release behaviour

379

For mechanistic evaluation the release profile can be used as a basic parameter. Although the most commonly

380

preferred release profile is zero order and the monophasic release is rarely observed. As a result of heterogeneous

381

degradation a bi-phasic or tri-phasic release profile may take place[48]. In case of surface coated PLGA nanoparticles

382

a biphasic release profile with a relatively rapid second phase is observed. The patterns observed in case of biphasic

383

drug release from PLGA nanoparticles are as ahead[49];

384

Phase I: Drug type, drug concentration and polymer hydrophobicity are some of the parameters which are associated

385

with an initial burst of drug release. As soon as the nanoparticle comes in contact with the dissolution medium, there is

386

a rapid penetration of water into the polymer matrix. the drug present on the surface on the nanoparticle is released as a

387

function of solubility. There is a random scission of PLGA molecule which results in a significant decrease in the

388

molecular weight of polymer. However, there is no appreciable weight loss and formation of soluble monomer product

389

during this phase.

390

Phase II: In the second phase, the thicker drug layer is depleted as a result of which the drug is released in a

391

progressive manner. Soluble oligomeric and monomeric products are formed due to the hydrolysis of the polymer

392

matrix. Hydrolysis results in creating a passage drug to be released by diffusion and erosion until complete polymer

393

solubilization takes place. However, in case of tri phasic release profile phase I is usually referred to as the burst

394

release. This kind of behaviour is attributed mainly to the hydration of non encapsulated drug particles present on the

395

surface or in close vicinity to the surface of the DDS. As degradation and hydration proceeds, polymer starts growing

396

dense, as a consequence of which, a slow release or diffusion of drug is observed in the Phase II. Burst release

397

sometimes is followed by erosion which is comparatively fast release phase and sometime is called as the second

398

burst.

399

9. Factors affecting degradation

400

The factors affecting degradation of PLGA nanoparticles are;

14 Page 14 of 27

401

(a) Polymer composition and molecular weight: The composition of the polymer plays a very decisive role in

402

determining the hydrophilicity and rate of degradation of any delivery matrix[50]. It has been deciphered that a

403

significant loss in the weight of the polymer is observed with a substantial increase in the polymer’s glycolic content.

404

The degradation rate of the polymer is directly affected by the amount of glycolic acid present and the degradation rate

405

increases with an increase in the glycolic acid proportion. Due to higher hydrophilicity there is a preferential

406

degradation of glycolic acid proportion as a consequence of which PLGA 50:50 (PLA:PGA) exhibited a faster

407

degradation than PLGA 65:35. Subsequently, PLGA 65:35 shows faster degradation than PLGA 75:25 and PLGA

408

85:15. Generally lower degradation rates are exhibited by high molecular weight polymers. The polymers having high

409

molecular weight possess longer polymeric chains and a longer duration is required for the degradation of these

410

polymers whereas on the contrary polymers having low molecular weight possess smaller polymeric chains hence

411

degradation of these polymers is achieved in a smaller duration.

412

(b) Drug type: Drug type also tends to play a very decisive role in deciding the mechanistic fate of polymer-drug

413

matrix degradation and drug release rate. The mechanism of degradation, as well as the rate of matrix degradation may

414

be changed from bulk erosion to surface degradation depending upon the type of encapsulated drug.

415

(d) Size and shape of the matrix: The degradation profile of large devices is shown to be significantly affected by the

416

ratio of surface area to volume. A faster and higher degradation of the matrix takes place with a higher surface area

417

ratio whereas, on the other hand it’s vice versa in case of matrix or devices having a smaller surface area to volume

418

ratio. Bulk degradation takes place faster than pure surface degradation resulting in a faster release of drug from the

419

devices with higher surface area to volume ratio.

420

(e) pH: It has been observed from in vitro studies that both, strongly alkaline and acidic media tends to accelerate the

421

polymer degradation. However, due to the autocatalysis of the polymer by the carboxylic end groups this difference

422

between the slightly acidic and neutral media becomes less pronounced[51].

423

(f) Drug load: The rate and duration of drug release is significantly affected by the amount of drug loading in the drug

424

delivery matrix. A larger initial burst release is possessed by matrices having higher drug content whereas, on the other

425

hand a comparatively smaller burst release is observed in case of matrices having lower content because of their

426

smaller polymer to drug ratio. Depending upon the type of drug, effect is attenuated when the drug content reaches a

427

certain level.

428

10. PLGA mediated drug delivery for cancer treatment

429

Cancer treatment via oral route is the most appealing approach till date because of its non invasive nature and

430

better patient compliance. However, due to its poor oral availability most of the anticancer drugs cannot be delivered

431

via oral route. When administered via oral route only a small fraction of the drug becomes available to the systematic

15 Page 15 of 27

432

circulation. For example, oral bioavailability of paclitaxel, docetaxel and doxorubicin have been found to be 1%,

433

< 10% and < 5%, respectively[52]. The underlying reason for this poor availability is the extensive first pass metabolic

434

effect by the cytochrome P-450 (liver microsomal enzyme)[44], as well as their efflux by an over-expressed plasma

435

membrane transporter P-gp efflux pump[53]. The P-gp is encoded via the gene MDR-1 which acts as an efflux pump.

436

After a prolonged chemotherapeutic sittings the efflux pump exports a wide range of chemo drugs and thus tends to

437

decrease the accumulation of functional drugs in MDR cells and hence body gradually stops responding to it, as a

438

result of which the therapeutic efficacy is depleted simultaneously followed by treatment failure[54]. The

439

administration of P-gp inhibitors in accordance to the drug of interest might prove to be an alternative in overcoming

440

this hindrance. On the contrary, P-gP administration is generally associated with aberrant toxicity, blocking of

441

physiological anticancer drug efflux from the normal cells and interrupted efflux of toxins from the body via the P-gP

442

efflux pump. The small intestine contains two types of cells i.e. enterocytes and M cells. Any liquid or soluble material

443

is absorbed in the small intestine via the enterocytes directly from the systematic circulation, while particulate matters

444

are absorbed by the M cells via lymphatics. In case of orally administered PLGA nanoparticles the absorption

445

predominantly takes place through the M cells present on the Payer's patches as well as via the isolated follicles of the

446

GALT. The efflux of drug by the P-gp efflux transporter can be effectively overcome by means of entrapping the

447

desired molecules within the voids of PLGA matrices. The drug absorbed from the enterocytes in the systematic

448

circulation tends to undergo first pass metabolism while, on the other hand, lymphatic absorption tends to bypass the

449

first pass metabolism[36]. Thus the incorporation of the active agents within the polymer matrix of the PLGA protects

450

the drug from getting depleted by the hostile environment of the gastrointestinal lumen. Small size and unique surface

451

chemistry of PLGA nanoparticles provided abilities like improved adhesion, absorption and transport of the drug. The

452

absorption pathway of a particulate delivery vehicle via M cells in Peyer’s patches. The encapsulation of chemo drugs

453

in the PLGA nanoparticles enabled them with an enhanced solubility, stability and an augmented drug

454

pharmokinetics[55]. The drug concentration can be controlled thereby reducing the risk of unwanted side effects and

455

allowing the useful treatment cycles to be maintained, without damaging the healthy cells.

456

11. Targeting strategies for efficient drug delivery

457

Chemo drugs possess many advantages but when administered inside the biological milieu they possess many

458

potential hazards viz. systemic toxicity, bone marrow suppression, cardiomyopathy and neurotoxicity. It is attributed

459

to the fact that chemo agents are unable to differentiate between normal and cancerous cells and the healthy cells or

460

tissues are damaged, limiting the maximal permissible dose of the drug. A large amount of drug has also to be

461

administered as some of the drug gets distributed into the non targeted organs and tissues ultimately leading to non

462

specific toxicity, followed by the rapid removal of the drug making the treatment process costly and uneconomical.

16 Page 16 of 27

463

With the implementation of nanoparticle mediated drug delivery systems these hurdles can be overcome[1]. In general,

464

nanoparticles mediated targeting can be achieved via two targeting strategies i.e. passive and active targeting;

465

(a) Passive targeting: The size of nanoparticles offers an additional advantage. They have the ability to extravasate

466

and accumulate inside the interstitial spaces, thus contributing to an enhanced permeability. Enhanced retention is

467

observed as a result of the ineffective lymphatic vessels, leading to an inefficient drainage of the tumour tissue [56].

468

Altogether these two phenomenon constitute the EPR effect which is considered to be a gold standard in designing the

469

effective anti cancer drug delivery system.

470

(b) Active targeting: In active targeting, the ligands are grafted at the surface of the nanoparticle. The tumour cells are

471

found to possess over expressed receptors and it is with these receptors that these ligands bind specifically. Improved

472

cellular internalization rather than an increased tumour accumulation has been found as the main reason for enhanced

473

anti tumoral efficacy of actively targeted nanoparticles. In case of active targeting two cellular targeting strategies has

474

been used;

475

(i) Targeting of cancer cells: Internalization prone cell surface receptors such as transferrin, folate, integrins or

476

EGFR etc. are mainly over expressed in cancer cells. This makes active targeting an alternative pathway for

477

improving the cellular uptake. Ligand mediated approach allows the cancer cells to be killed directly followed by

478

the generation of cytotoxicity against the cells which are present at the periphery of the tumour [57].

479

(ii) Targeting of tumour endothelium: Recognition of specific receptors such as VEGFR-1 and VEGFR-2, the

480

integrins (αvβ3, α5β), VCAM-1 or the MMPs by targeting ligands helps in an effective targeting of the tumour

481

endothelium[1]. Folkman et al. was the first to propose the rationale behind this targeting. It has been suggested

482

that tumour growth might be inhibited by preventing angiogenesis[58]. Due to lack of oxygen the endothelium in

483

tumour cells is demolished ultimately leading to the death of tumour cells. However, the size and metastatic

484

capabilities of tumours can be restrained by assailing the surge of blood supply. The tumour core comprises of

485

angiogenic blood vessels, which in turn support the sustenance of the tumour cells. The nanoparticle bind with

486

these angiogenic blood vessels and kill them, resulting in an indirectly killing of the tumour cells. Still, the

487

nanoparticle mediated approach possess many advantages[8];

488

(i) In order to direct these nanoparticles to the target site, no extravagation of nano-carrier is

489 490 491 492 493

required (ii)

When

administered

intravenously,

these

particles

tends

to

bind

quickly

to

their

receptor

sites (iii) The possible risk of emerging resistance is avoided as the endothelial cells are genetically more stable than the tumour cells

17 Page 17 of 27

494 495 496

(iv) The majority of endothelial cells are expressed in almost all types of tumour, hence making this approach ubiquitous. 12. Ligand anchored PLGA nanoparticle for cancer therapy

497

Cancer cells unlike normal cells have an innate tendency to proliferate rapidly and this rapid proliferation or growth

498

is supported by some over exposed receptors present on the surface of the tumour cell. This receptor allows the uptake

499

of growth factor via receptor mediated endocytosis. This mechanism can be used as a Trojan horse for site specific

500

delivery of anticancer agents. The surface of the nanoparticles are decorated with ligands such as antibodies that tend

501

to bind specifically with these receptors[36]. The desired ligands can be attached to the surface of the nanoparticles via

502

simple physical associations or conjugation reactions.

503

Carbodiimide chemistry is most commonly used for conjugation reactions. Nanoparticles can be surface derivatized

504

using conjugation reaction. A water soluble carbodiimide reagent such as EDC/DCC is allowed to react with a

505

carboxyl group present in the PLGA, leading to the formation of an amine reactive O-acylisourea intermediate. This

506

intermediate reacts with the amine group present in the ligand, resulting in the formation of PLGA-ligand conjugate.

507

However, this reaction is also prone to some other unwanted secondary reaction. In order to avoid any further reaction

508

NHS is added into the reaction mixture and the amine reactive intermediate so formed earlier gets transformed into a

509

NHS-ester derivative. The ester derivative immediately reacts with any primary amine group present in the reaction

510

mixture, along with the liberation of NHS and ultimately leaving behind PLGA-ligand conjugate. Importantly, the

511

conjugation reaction should proceed in an aqueous environment to some portions of the free carboxylic end group

512

remains embedded in the PLGA nanoparticle, resulting in limited availability for direct conjugation. To overcome this

513

situation, PLGA is dissolved in an organic solvent such as dimethyl formamide, prior to the conjugation reaction [59].

514

The harsh conjugation steps can be avoided by using some other ligands such as non covalent binding of Biotin-PEG-

515

NH2 with an Avidin functionalized PLGA nanoparticle. Ligands such as antibodies and Fab fragments can be attached

516

to PLGA nanoparticles.

517

Substantial research has been focused on ligand mediated PLGA nanoparticles, but there are limitations which have

518

hampered the use of these engineered nanoparticles in practical situations. In vivo studies have revealed that a

519

significant amount of injected dose gets accumulated in the number of RES organs such as liver, spleen etc. This is

520

undesirable as the anticancer drugs may damage the MPS organs. The clearance time of these nanoparticles is

521

expedited because of the recognition of these targeted nanoparticles by the MPS. Despite of the fact that ligand

522

targeted approach possess several limitations, ligand targeted nanoparticles have shown an enhancement in the

523

anticancer effect of the entrapped drug by facilitating the cellular uptake and intracellular retention of the nanodrug

18 Page 18 of 27

524

carriers, which in turn has augmented their anti tumour efficacy [36]. A comprehensive overview of different ligands

525

anchored or conjugated to PLGA nanoparticles is provided in Table 2[60-62].

526

13. PLGA nanoparticles as thriving mediator

527

(a) Gene delivery for cancer treatment: In the current scenario of anti cancer field, gene therapy is considered as one

528

of the most promising tool for targeted drug delivery. This technique can be utilized for treating a variety of infectious

529

diseases such as monogenic diseases and cancer [63]. On its way from outside of the cells to the cellular milieu a gene

530

or macromolecule has to face many hurdles such as poor permeability, membrane non- selectivity and degradation in

531

the endo-lysosomal environment. In order to circumvent these obstacles non viral novel vectors have appeared as a

532

new promising approach.

533

PLGA nanoparticles have been explored as a delivery vehicle for DNA, so that the target gene is effortlessly

534

transfected with the cancer cells. For this purpose, the surface properties of PLGA nanoparticles such as surface charge

535

or coating have to be appropriately tailored to make them an efficient carrier for DNA transfer into cancer cells [36].

536

Gene silencing via siRNA is currently the fastest growing sector of the antigene field for target validation and

537

therapeutic applications. However, systematic deliverability of siRNA into target cells is often challenged by their

538

rapid degradation and poor cell penetration properties. In order to overcome these problems, a broad spectrum of viral

539

and non viral vectors has been successfully utilized to improve the targeted delivery of siRNA to cancer cells and

540

protect them from premature degradation in the biological milieu. PLGA based systems have also been investigated for

541

the targeted delivery of siRNA to cancer cells and to induce gene silencing. Studies have been carried out for the

542

delivery of DNA or a specific gene via PLGA nanoparticles as tabulated in Table 3[64-66].

543

(b) Diagnosis and imaging of cancer: In the field of clinical oncology, tumour imaging plays a very vital role as it

544

helps in determining the recurrence of the solid tumours along with the monitoring of the therapeutic responses.

545

Currently available clinical diagnostic methods are unable to detect the cancer in early stages [1]. However, the

546

development of a non invasive molecular imaging system might allow the detection of tumour at an early stage. Recent

547

advancements in nanoparticles have facilitated the use of the contrasting agents for imaging [23]. Wang et al. prepared

548

supramagnetic iron oxide loaded PLGA nanoparticles for MRI [67]. The imaging effects are enhanced along with an

549

increase in the half life of nanoparticles in the blood stream with reduced severe side effects. In a different experiment

550

by Acharya et al. sentinel lymph node of wistar rat has been detected by encapsulating a radiotracer, 99mTC in PLGA

551

nanoparticles [23].

552

(c) Therganostics of cancer: Therganostics collectively describes the therapeutic and diagnostic agents. It may

553

combine passive and active targeting, environmentally responsive drug release, molecular imaging and other

554

therapeutic functions under a single platform [68]. For example, magnetic nanoparticles containing doxorubicin were

19 Page 19 of 27

555

further encapsulated in PLGA nanoparticles through hydrophobic interactions. In another study, PLGA containing

556

magnetic nanoparticle are designed for serving the dual function of drug delivery and imaging[69]. MRI scans reveals

557

that a better contrast is presented by these nanoparticles in comparison to commercially available contrasting agents.

558

14. Pitfalls encountered with PLGA nanoparticle based drug delivery system

559

Apart from developmental and nanoethical aspects, there are obstacles that may be encountered with PLGA

560

nanoparticle based drug delivery system.

561

(i)

One of the major pitfalls relates to the fact that the EPR is often misunderstood. It is a phenomenon which is

562

heterogeneous in nature varying substantially from model to model, and from patient to patient. Potential

563

usefulness of active drug targeting is also overestimated at times which becomes another major aspect. On

564

theoretical basis, it can be said that targeted nanoparticles must be retained more efficiently and rapidly than non

565

targeted ones. However, an increase in immunogenicity and protein adsorption often takes place along with the

566

introduction of targeting moieties[56].

567

(ii)

Although PLGA based nanoparticles offers advantage like high encapsulation efficiency in comparison to other

568

nanoparticle based DDS, still PLGA based nanoparticles are accompanied with poor drug loading. This poor

569

drug loading might prove to be problematic for some drugs in the designing of PLGA nanoparticles[8].

570 571

(iii) High burst release rate of drug from nanoparticles is often accompanied with the drug delivery system. Thus, the efficacy of the drug delivery system is lost followed by a non targeted delivery of the payload or drug.

572

(iv) The degradation of PLGA polymer leads to the formation of acids resulting in hampering the therapeutic

573

application. If the encapsulant is pH sensitive, it might not become feasible to be delivered via PLGA

574

nanoparticles.

575

(v) The local tissue reactions occur at the site of application and it becomes another area of concern. Studies have

576

also suggested that specific biodistribution and toxicological profiles also get created when nanoparticle of any

577

material is being used for therapeutic application[70].

578 579 580 581 582

(vi) Relatively poor drug loading, higher cost of production and difficulty in scaling up are some of the other prominent hurdles limiting the use of PLGA nanoparticles in clinical trials. (vii) A thorough and fundamental knowledge about the effect of various process parameters on the preparation of nanoparticles is still limited[27]. (viii) The particle size and morphology of the nanoparticles are affected by two major factors viz.,

583

homogenization

584

of these parameters is still lacking.

conditions

and

droplet

size

distribution.

A

thorough

understanding

20 Page 20 of 27

585

(ix)

586

Heterogeneous expression levels of the targeted receptor in cancer cells also become an obstacle during in vivo application. Various physiological barriers prevent these nanoparticles from reaching the target tissues.

587

(x)

The complete characterization of in vivo behaviour of these nanoparticles is still lacking[59].

588

(xi)

Complexity of drug release from PLGA based drug delivery system makes it difficult to generalize the results

589 590

obtained with specific DDS[44]. 15. Conclusion

591

Despite of the fact that PLGA nanoparticles based drug delivery system possesses a number of advantages, a

592

paradoxical prospective is represented by PLGA based drug delivery systems. The relatively low drug loading

593

efficiency is the major hurdle limiting the use of drug-loaded PLGA nanoparticles in clinical trials. Preparation of

594

polymeric nanoparticle is a state-of-art technology requiring a suitable protocol, thorough utility of homogenization

595

process, appropriate surfactants and creditable co-surfactant to obtain desired polymeric nanoparticles with optimum

596

property enhancement. It is required to focus the line of future research on developing the techniques that can provide

597

precise control over the particle size and morphology, which are considered to be the determining factors for

598

applications of these wondrous particles. Many efforts have still to be performed to better control nanoparticles size,

599

polydispersity, charge surface and to render these features easily reproducible during synthesis steps. The challenges

600

encountered with PLGA nanoparticles can be overcome through an extensive and thorough evaluation of the

601

pharmacokinetics, biodistribution and toxicity. The PLGA degradation and the drug release rate can be accelerated by

602

greater hydrophilicity, increase in chemical interactions among the hydrolytic groups, less crystallinity and larger

603

volume to surface ratio of the device. All of these factors should be taken into consideration in order to tune the

604

degradation and drug release mechanism for desired application. Proper selection of preparative method,

605

formulation/reaction variables and appropriate scale-up techniques/methods, combined with coordinated efforts from

606

the industrial and academic sectors, can lead to successful commercialization of engineered PLGA nanoparticles.

607

Nevertheless, the future remains exciting and wide open, and further advances are required to turn the concept of drug

608

loaded PLGA NP technology into a realistic practical application as the next generation of drug-delivery systems.

609

Acknowledgement

610 611

Authors are thankful to DST, Govt. of India, for providing financial support to IFSC through PURSE grant. 16. References

612

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[2] Y. Liu, H. Miyoshi, M. Nakamura, Nanomedicine for drug delivery and imaging: a promising avenue for cancer

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[3] S.A. Wickline, A.M. Neubauer, P.M. Winter, S.D. Caruthers, G.M. Lanza, Molecular imaging and therapy of

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[12] S.S. Acharya, S. K., PLGA nanoparticles containing various anticancer agents and tumour delivery by EPR effect,

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[13] S.J. Siegel, J.B. Kahn, K. Metzger, K.I. Winey, K. Werner, N. Dan, Effect of drug type on the degradation rate of

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PLGA matrices, Eur J Pharm Biopharm, 64 (2006) 287-293.

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[14] N. Passerini, D.Q. Craig, An investigation into the effects of residual water on the glass transition temperature of

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polylactide microspheres using modulated temperature DSC, J Control Release, 73 (2001) 111-115.

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[15] Z. Panagi, A. Beletsi, G. Evangelatos, E. Livaniou, D.S. Ithakissios, K. Avgoustakis, Effect of dose on the

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biodistribution and pharmacokinetics of PLGA and PLGA-mPEG nanoparticles, Int J Pharm, 221 (2001) 143-152.

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[16] R.A. Jain, The manufacturing techniques of various drug loaded biodegradable poly(lactide-co-glycolide) (PLGA)

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devices, Biomaterials, 21 (2000) 2475-2490.

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[17] S. Mao, J. Xu, C. C;, O. Germershaus, Effect of WOW process parameters on morphology and burst release of

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FITC-dextran loaded PLGA microspheres, Int.J.Pharm, 334 (2007) 137-148.

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[18] P.T.G. Hua. F.J, Lee, D.S., facile preparation of highly interconnected macroporous PLGA scaffolds by liquid-

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[19] D.S. D’Mello SR, Das NG., Polymeric Nanoparticles for Small-Molecule Drugs: Biodegradation of Polymers and

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Fabrication of Nanoparticles. Drug Delivery Nanoparticles Formulation and Characterization, (2006).

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[20] G. Lambert, E. Fattal, P. Couvreur, Nanoparticulate systems for the delivery of antisense oligonucleotides, Adv

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FIGURE CAPTIONS

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Fig.1(a). Direct Conjugation of PEG to surface of premade PLGA nanopaticles

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Fig.1(b). Activated Conjugation of PLGA to heterofunctional PEG

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Fig.1(c). Ring opening polymerization of lactide and glycolide

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Figure 1(a)

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Figure1(b)

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Figure 1(c)

774 775 776 777

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LIST OF TABLES Table1. PLGA-PEG nanoparticles for anti cancer drug delivery

Nanoparticles

Drug loaded

Target

Inference

Reference

PLGA-PEG nanoparticles

Paclitaxel

HeLa

Entrapment of paclitaxel within a PEGylated PLGA nanoparticle resulted in higher tumor growth inhibition, followed by a significant augmentation in the tumor specific localization of the drug

[40]

PLGA-mPEG nanoparticles

Cisplatin

BALB/c mice

Higher survival rate and delayed tumor growth was observed for PLGA-mPEG nanoparticle treated mice in comparison with the animals treated with the free drug

[41]

PLGA-PEG nanoparticles

Docetaxel

Solid tumors

Biological half life of the drug is significantly augmented, while imparting considerable solid tumor accumulation

[42]

Table 2 Ligand targeted PLGA nanoparticles for cancer therapeutics

Nanoparticle

Drug loaded

Ligand

Target site

Inference

Reference

PLGA

Paclitaxel

ScFv antibody

Hepatic cancer

Improved cellular cytotoxicity against Chepp-3 cells

[60]

PLGA-PEG

Cisplatin

PSMA targeting aptamer

Prostate

The NPs were readily taken up by receptor mediated endocytosis

[61]

PLGA-PEG

Paclitaxel

RGDp

Human umbilical vein endothelial cells

Enhanced uptake by integrin expressing malignant cells

[62]

781 782

26 Page 26 of 27

783 784 785

Table 3 PLGA nanoparticles for gene delivery

Nanoparticles

Gene Delivered

Target

Inference

PLGA nanoparticles

DNA

COS-7 and Cf2th cells

Localization into the endolysosomal compartment, 250 fold protein expression in cells

Chitosan-PLGA nanoparticles

Antisense oligonucleotides DNA/RNA

Lung cancer cells

Efficient delivery of antisense oligonucleotides

DEPA-PVAPLGA nanoparticles

Anti-luciferase siRNA

H1299 cell line

80%-90% knockdown of the luciferase reporter gene

Reference [64]

[65]

[66]

786 787 788

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