Engineered PLGA Nanoparticles

Critical Reviews™ in Therapeutic Drug Carrier Systems, 28(1), 1–45 (2011)

Engineered PLGA Nanoparticles: An Emerging Delivery Tool in Cancer Therapeutics Amit K. Jain, Manasmita Das, Nitin K. Swarnakar, & Sanyog Jain* Centre for Pharmaceutical Nanotechnology, Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), SAS Nagar (Mohali), Punjab, India *Address all correspondence to Sanyog Jain, Centre for Pharmaceutical Nanotechnology, Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, SAS Nagar (Mohali) Punjab 160062, India; Tel.: 91-172-2292055; Fax: 91-172 2214692; [email protected] or [email protected].

ABSTRACT: Nanocarriers formulated with the US Food and Drug Administration-approved biocompatible and biodegradable polymer poly(lactic-co-glycolic acid) (PLGA) are being widely explored for the controlled delivery of therapeutic drugs, proteins, peptides, oligonucleotides, and genes. Surface functionalization of PLGA nanoparticles has paved the way to a variety of engineered PLGA-based nanocarriers, which, depending on reticular requirements, can demonstrate a wide variety of combined properties and functions such as prolonged residence time in blood circulation, enhanced oral bioavailability, site-specific drug delivery, and tailored release characteristics. The present review highlights the recent leaps in PLGA-based nanotechnology with a particular focus on cancer therapeutics. Starting with a brief introduction to cancer nanotechnology, we then discuss developmental aspects and the in vitro and in vivo efficacy of PLGA-based nanocarriers in terms of targeted drug or gene delivery. The main objective of this review is to convey information about the state of art and to critically address the limitations and the need for further progress and clinical developments in this emerging technology. KEY WORDS: PLGA nanoparticles; cancer therapy; cancer targeting

I. INTRODUCTION Cancer refers to a complex disease in which a group of cells display uncontrolled growth or proliferation, followed by invasion and metastasis. These three malignant features differentiate cancer from benign tumors, which are self-limited and do not metastasize. Cancer is the second leading cause of death worldwide. According to a recent survey by American Cancer Society, 562,340 people in the United States died out of cancer in 2009. The apparent impenetrability of the disease is largely attributed to the multiple, often redundant pathways that evolve through the genetic instability of cancer cells. Although the 21st century has witnessed astounding progress in the area of fundamental cancer biology, drug development, and molecular imaging, cure of cancer in many cases remains beyond the grasp of physicians. Over the last two decades, several approaches have been used to treat different forms of cancer. A thorough discussion of these is beyond the scope of this review, and readers are directed elsewhere for more detailed information.1,2 Briefly, among the various therapeutic strategies, chemoprevention has been used the most

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effectively to treat cancer. In fact, many high-quality clinical trials recommend the use of chemoprevention in defined circumstances.3 For example, selective estrogen receptor modulators such as tamoxifen and raloxifene have been extensively used to prevent breast cancer in high-risk women.4 Likewise, a 5-alpha reductase inhibitor, finasteride, has been shown to lower the risk of prostate cancer.5 Similarly, COX-2 inhibitors such as rofecoxib and celecoxib (Celebrex) have been approved by US Food and Drug Administration (FDA) for the treatment of familial adenomatous polyposis.6 However, a major problem associated with conventional therapies is that they are indiscriminate; although they manage to kill the rapidly proliferating cells, such as tumor cells, they also affect normal cells. Therefore, there is a crucial need for the development of a more effective cancer therapy that not only minimizes side effects but also directly targets diseased cells. “Cancer nanotechnology” is a fascinating area of nanotechnology that has emerged in the form of a variety of nanocarriers.7 This technology holds tremendous potential to solve several limitations of conventional drug-delivery systems, such as nonspecific biodistribution and targeting, lack of water solubility, poor oral bioavailability, and low chemotherapeutic indices. Nanostructured materials possess the unique ability to perform dimensionally relevant interactions with biological entities, either on their surface or inside of cells. By gaining admittance to so many areas of the body, they have the potential to detect disease and deliver treatment in a variety of ways. Cancer nanotechnology involves the creation, formulation, and development of different physical systems having a size in the range of 1 to 1000 nm. These nanoscale materials possess unique optical, electronic, and structural properties that are not available from either individual molecules or bulk solids. These properties can be successfully exploited for the construction of new tools or devices that can effectively track and target cell surface receptors or molecules specific to cancer.7 Drug-loaded nanocarriers, combined with the identification of cancer-specific molecular targets, can provide better cellular internalization so that the side effects associated with anticancer drugs can be significantly reduced. The current focus in pharmaceuticals is gradually shifting toward a “smart drug” paradigm, in which increased efficacy and decreased toxicity are the main motivating factors. Nanotechnology offers a “smarter” and less invasive alternative to conventional therapeutic cocktails (surgical intervention, chemotherapy, radiation, and surgery), thereby enhancing the expectancy and quality of life for the individual suffering from this disease.8 Over the last decade, substantial effort has been devoted to the design and development of nanometer-sized targeted probes for cancer diagnosis and therapy. Recent advancements in nanotechnology have fostered the development of “smart” nanostructures in the form of colloidal drug-delivery systems that, depending on reticular requirements, can demonstrate a wide variety of combined properties and functions. These include: i) increased stability in the blood circulation; ii) the ability to accumulate specifically or nonspecifically in the pathological zone of interest; iii) high specific internalization by the target cells compared with normal cells; and iv) an effective, intracellular drug-delivery and release mechanism after reaching the target site. As suggested by different reports, surface-engineered nanocarriers loaded with bioactives offer an array of advantages over free bioactives.9 Physical

Critical Reviews™ in Therapeutic Drug Carrier Systems

Engineered PLGA Nanoparticles in Cancer Therapeutics

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entrapment or chemical conjugation of the therapeutic drug with such tailored nanocarriers protects the drug from premature degradation in the biological milieu, enhances its tissue-specific absorption and accumulation, and controls its pharmacokinetics and biodistribution profile with concomitant improvement of intracellular penetration. Considering the unique advantages of drug-loaded/conjugated nanocarriers over free drugs, a variety of tailored nanocarriers with different material properties have been extensively investigated for cancer diagnosis and therapy. These include polymeric drug conjugates10; polyethylene glycol-containing (PEGylated) and non-PEGylated liposomes11; polymerosomes12-14; micelles (lipid-based and polymeric)15,16; gold silica nano-shells17; gold nanoparticles18; immuno-PEG liposomes19; immunoliposomes20; immunotoxins, immunopolymers, and fusion proteins21,22; polymeric nanoparticles23,24; and carbon nanotubes.25,26 Among this broad spectrum of functional nanomaterials, nanoparticles consisting of synthetic, biodegradable polymers have been extensively used for the construction of delivery devices for therapeutic drugs, genes, and molecular imaging agents due to their excellent biocompatibility and biodegradability. Poly(lactic-co-glycolic acid) (PLGA) is one of the most successfully used biodegradable nanosystems in the nanomedicine arena because it can easily hydrolyze in the body to produce the nontoxic, biodegradable metabolite monomers lactic acid and glycolic acid. In view of its excellent biocompatibility and biodegradability, PLGA has been approved by the FDA for drug delivery. A number of articles have reviewed the different aspects of PLGA-based micro- and nanotechnology. Mundargi et al.27 outlined the research and developmental activities on nano-/microparticle-based delivery systems of PLGA and PLGA-derived polymers with regard to macromolecular therapeutics. Lu et al.28 reviewed the application of PLGAbased nanotechnologies and tools in the diagnosis and treatment of cardiovascular disease, cancer, and immunology, and in vaccines and other diseases and devices. More recently, Avgoustakis29 reviewed the preparation, properties, and potential applications of PEG-conjugated PLGA (PLGA-PEG) nanoparticles for the intravenous and mucosal delivery of anticancer agents, proteins, oligonucleotides, and genes. While most of these articles address the general aspects of PLGA-based nanotechnology in drug delivery and relevant applications, this review article is designed to provide the readers with thorough, updated information on the developmental aspects as well as the applications of engineered PLGA nanoparticles in cancer therapeutics. Starting with a brief introduction on cancer nanotechnology, we then discuss the design, synthesis, and therapeutic application of engineered PLGA-nanoparticles with special emphasis on ligand-decorated PLGA nanocarriers. The main objective of this review is to convey information about the state of art and to critically address the limitations and the need for further progress and clinical developments in this emerging technology. II. TARGETED DELIVERY OF CHEMOTHERAPEUTICS: PASSIVE AND ACTIVE TARGETING STRATEGIES As stated above, conventional cancer therapy usually suffers from drawbacks that can be overcome by the application of nanotechnological concepts. Nanoparticulate sys-

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tems provide a high degree of site selectivity, which is a prerequisite to avoiding the unwanted side effects caused by the majority of anticancer drugs. Different nanoparticulate systems have been investigated and envisaged for cancer therapeutics. Any nanoparticulate system that is administered via the intravenous route goes primarily into the liver and the mononuclear phagocyte system (MPS) by virtue of the body’s natural defense mechanism. Tumor vasculature is highly leaky in nature compared with the normal endothelial lining of blood vessels. Nanocarriers exploit such specific characteristics of tumors, such as leaky blood vessels, defective vasculature architecture, deprived lymphatic drainage, and limited diffusion volume (2 mm3) to gain access to the tumor interstitium.8,30 The combination of leaky vasculature and poor lymphatic drainage results in what is known as the enhanced permeation and retention effect.31 Size is a critical issue dictating the fate of nanocarriers in vivo. Nanoparticles larger than 200 nm are sequestered by phagocytotic cells of the spleen, while particles smaller than 5 nm are rapidly removed through renal clearance. Particles escaping filtration are subject to opsonization, resulting in recognition and clearance by Kupffer cells and other tissue macrophages. Surface modification of nanocarriers with hydrophilic polymers such as PEG improves their accessibility to the tumor site by prolonging their residence time in blood circulation, thus causing less interaction with serum opsonins (i.e., less recognition by the reticuloendothelial system, RES).32 Other than the enhanced permeation and retention effect, intracellular uptake of nanoparticles is also facilitated by introducing positive or negative charges on the surface of nanoparticles.24 While positively charged nanocarriers bind to cells through electrostatic interactions with the negatively charged cell membranes, endocytosis of negatively charged nanoparticles occurs through both protein-mediated phagocytosis and diffusion. However, it should be noted that the passive targeting of nanocarriers depends on the degree of tumor vascularization and angiogenesis. Moreover, this effect may not be achieved in all tumors because the porosity and pore size of tumor vessels vary with the type and status of the tumor.33 Furthermore, the elevated interstitial fluid pressure, a common condition prevailing in most solid tumors, might inhibit the efficient uptake and homogeneous distribution of nanoparticles and/or drugs in tumor tissues. Thus, to prevail over these limitations of passive targeting and unwanted access of drugs to different sites, a variety of targeting ligands can be anchored to nanocarriers by applying diverse conjugation chemistry. Such ligand-anchored nanocarriers can specifically recognize and adhere to biomarkers overexpressed on the surface of malignant cells via a ligand-receptor binding interaction. This strategy, known as active targeting, provides an effective means to target and deliver drugs to a specific place in the body, thereby reducing the systematic side effects associated with nonspecific targeting and biodistribution of the chemotherapeutic drug. Targeting agents can be broadly classified as proteins (e.g., antibodies and their fragments), receptor ligands (e.g., peptides, vitamins, and carbohydrates), and nucleic acids (aptamers). The active targeting strategy exploiting different tumor-specific ligands has been summarized in Figure 1.



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FIGURE 1. Ligands for active targeting in cancer.

III. TARGETED DRUG DELIVERY USING PLGA NANOPARTICLES III.A. Simple Drug-loaded PLGA Nanoparticles PLGA is an FDA0approved, biocompatible, biodegradable, and safely administered polymer34 that is widely employed in a variety of biomedical applications ranging from surgical tissue grafting to ligament reconstruction.35 In addition to having excellent biocompatibility, PLGA has favorable degradation kinetics35 that depend on the properties summarized in Figure 2. PLGA nanoparticles have been widely employed for the loading or encapsulation of a variety of anticancer drugs, ranging from hydrophilic drugs such as doxorubicin,36 5-flurouracil,37 and cisplatinu38 to hydrophobic drugs such as paclitaxel.39 A variety of methods have been employed to prepare drug-loaded PLGAbased nanoformulations. A brief summary of these methods is provided in Table 1.

FIGURE 2. Factors affecting the degradation of PLGA. (Modified from Bala et al., 200435 Used with permission.)


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TABLE 1 . Methods of Preparing PLGA Nanoparticles Method of Preparation

Drug(s) Incorporated

Brief Description

Remarks

Emulsionevaporation

Drug and polymer dissolved in organic solvents and emulsified in aqueous solution of emulsifier/stabilizer. The resulting emulsion droplet size is reduced by high-energy source either by sonication and homogenization. Then organic phase is evaporated under reduced pressure. The nanoparticles are collected by ultracentrifugation and freeze-dried.146

Paclitaxel39,148

Particle size can be controlled by stirring rate and other conditions148; high efficiency for lipophilic drugs.39

Double-emulsion evaporation

Addition of aqueous drug solution to the polymer solution under vigorous stirring conditions to form a water/oil emulsion, which is added into the stabilizer solution with stirring to form a water/oil/water emulsion. The resulting double emulsion is subjected to solvent evaporation.149

Cisplatin38

Optimization parameters include percentage of drug loading, concentration of stabilizer, polymer concentration, and volume of external phase.

Salting-out

Solution of polymer and drug in acetone is added to aqueous solution contacting the stabilizers and salting-out reagent under mechanical stirring. Emulsion is diluted with water, which leads to the diffusion of acetone. Cross-flow filtration is employed to remove the acetone and salting-out reagent.151

9-Nitrocamptothecin152

Optimization parameters include stirring rate, concentration of stabilizer, polymer concentration, and type of electrolyte concentration.

Emulsificationdiffusion

Addition of polymer solution in a partially water-miscible solvent such as ethyl acetate, benzyl alcohol, or propylene carbonate, which is added to the stabilizer solution under stirring conditions. Water is added to the system, which leads to destabilization. The solvent diffuses into the external phase and is ultimately evaporated, leading to the formation of nanoparticles.147

--

Provides more intensive purification step, suffers from the lower entrapment efficiency of hydrophilic drugs, increased by addition of medium-chain glyceride into the aqueous phase.

Solvent displacement/ nanoprecipitation

Solution of polymer, drug, and lipophilic surfactant in semipolar solvent such as acetone and ethanol injected into aqueous containing the stabilizers under magnetic stirring. Nanoparticles are formed by rapid solvent diffusion, and the solvent is removed under the reduced pressure.37

Paclitaxel,59 docetaxel103,148,153

Particle size is affected by the injection rate of organic solvent into aqueous solution; narrow particle size for different formulations.



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Entrapment of hydrophobic anticancer drugs into PLGA-nanoparticles is mainly accomplished via the emulsion-diffusion-evaporation method.40 Optimization of different formulation variables is a prerequisite for producing drug-loaded nanoparticles with maximum entrapment efficiency and desired particle size. These qualities depend on: i) the droplet size of the emulsion, ii) the nature of the organic phase, iii) the ratio of the organic to the aqueous phase, and iv) the surfactant/stabilizer used during the preparative process. The choice of method for droplet size reduction (i.e., homogenization or sonication) influences the particle size and polydispersity index (PDI) of nanoparticles. Usually, nanoparticles formed by the sonication method are smaller with better PDI. For example, we prepared tamoxifen-loaded PLGA nanoparticles and compared the efficiency of the two methods for droplet size reduction. Compared with homogenization, particles with small size and better PDI were produced via sonication.41 The choice of the surfactant is also crucial because it affects the surface charge, particle size, and entrapment efficiency of PLGA nanoparticles.41,42 Although it is obvious that the hydrophobic nature of PLGA will favor the entrapment of hydrophobic drugs into the hydrophobic polymeric core, the extent of drug entrapment is often influenced by the nature of the surfactant used. We observed lower entrapment efficiency of tamoxifen in PLGA nanoparticles when the surfactant was changed from polyvinyl alcohol (PVA) to didodecyldimethylammonium bromide (DMAB) while maintaining all other parameters constant.41 The lower drug entrapment in presence of DMAB may be attributed to the higher solubility of drug in the surfactant solution compared with its affinity toward the polymer phase, which suggests that the solubility of drug in the surfactant solution should be thoroughly optimized prior to the formulation of nanoparticles. It is also interesting that these formulation variables not only influence the drug encapsulation and size of the particles, but also their release characteristics and in turn their cytotoxicity profile. For example, Dong and Feng43 employed high-pressure homogenization to prepare PLGA nanoparticles for controlled release of paclitaxel. A biphasic profile of paclitaxel was obtained with a fast release rate in the first 3 days, followed by a slow first-order release. The drug-loaded nanoparticles showed higher or comparable cytotoxicity against glioma C6 cells compared with the free drug, Taxol. The effective internalization of fluorescent coumarin-6-loaded nanoparticles was also shown by confocal laser scanning microscopy.43 In another study, Mo and Lim enhanced the efficiency of paclitaxel-loaded PLGA nanoparticles by carrying out the synthesis in presence of a release modifier, isopropyl myristate, and conjugating these drug-loaded nanoparticles with wheat germ agglutinin. In vitro cytotoxicity studies against the adenocarcinomic A549 and H1299 cell lines corroborated that wheat germ agglutinin-conjugated nanoparticles had superior antiproliferation activity compared with conventional paclitaxel formulations. In vivo studies further established that the growth of A549 tumor nodules was inhibited without inducing a significant weight loss in SCID (severe combined immunodeficiency) mice. The enhanced efficacy of wheat germ agglutinin-conjugated nanoparticles over conventional paclitaxel formulation was attributed to the more efficient intracellular localization of paclitaxel via wheat germ agglutinin-receptor-mediated endocytosis and isopropyl myristate-facilitated intracellular release of paclitaxel.44


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While the emulsion-diffusion-evaporation method is the primary choice for entrapment of hydrophobic drugs into PLGA-based nanocarriers, a double-emulsion method is usually employed for the formulation of PLGA nanoparticles with hydrophilic drugs.45 The double-emulsion method usually leads to lower entrapment efficiency compared with the emulsion-diffusion-evaporation method. Using the double-emulsion method, the entrapment efficiency of drugs can be increased by the saturation of internal phase or the outer phase with the drug itself.46 Except for the ratio of the internal aqueous phase to the organic phase, the optimization parameters for the double-emulsion method remain the same as for the solvent-evaporation-diffusion method, and this additional parameter needs to be optimized. Chicken cystatin, a model protein inhibitor of cysteine proteases, was entrapped in PLGA nanoparticles by Cegnar et al.47 A low-energy emulsification method was employed by these researchers for preserving the protein integrity in the PLGA nanoparticles. Another hydrophilic drug, doxorubicin, was entrapped in PLGA nanoparticles by Kalaria et al. using the same method.36 Moreno et al. loaded cisplatin into PLGA nanoparticles using the double-emulsion method and compared its properties with the PLGA microparticles prepared by same method. In addition, we prepared PLGA microparticles using a different protocol: the double-solvent-evaporation method. Similar entrapment efficiency was found in both microparticles and nanoparticles, but a greater amount of drug was loaded into the microparticles. The antiproliferative effect of cisplatin was found to be dependent on the initial burst effect of cisplatin. A duality pattern was observed in the cell-cycle distribution using both formulations. Compared with free cisplatin, both formulations showed rapid inhibition of cells growth, followed by a significant loss of cells in the G0/G1 and G2/M phases, followed by an increase in the number of cells in the sub-G phase, and it was suggested that the controlled-release formulations of cisplatin were able to induce a more effective apoptosis than free cisplatin48 (Figure 3). Another method for the formulation of anticancer drug-loaded PLGA nanoparticles is the solvent displacement or nanoprecipitation method. This method is employed for the encapsulation of hydrophobic and hydrophilic drugs in PLGA nanoparticles.49 It is a convenient, reproducible, fast, and economic method for the one-step synthesis of PLGA nanoparticles with narrow size distribution.50 The particle size and entrapment efficiency in the nanoprecipitation method depends upon the type of water-miscible solvent used (e.g., acetone and ethanol) and its rate of addition into the aqueous medium. The entrapment efficiency of the drug in PLGA nanoparticles prepared by nanoprecipitation method could be increased by varying any one of the following factors: i) the pH of the aqueous phase, ii) the salt form of the drug, or iii) the addition of polymeric excipients to the outer phase.51 Betancourt et al. investigated the entrapment of doxorubicin in acid-capped PLGA nanoparticles using the nanoprecipitation method with bovine serum albumin as the stabilizer, and achieved an approximately 5% loading efficiency. A pH-dependent release of drug was obtained due to accelerated degradation of the polymer and decreasing ionic interaction between the drug and the polymer at acidic pH. Moreover, Betancourt et al. demonstrated the high efficiency of doxorubicin-loaded nanoparticles against MDAMB-231 breast cancer compared with the free drug.52



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FIGURE 3. (A) SEM image of microparticles. (B) SEM image of nanoparticles. (C) In vitro release profiles of cisplatin (means ± SD; n = 3). Release profile over the first few time points have been presented as inset. (D) Effect of different doses and exposure times of cisplatin in solution or encapsulated on cell proliferation. Free cisplatin treatments (•), nanoparticles (▲), and microparticles (⌂). (E) Cell-cycle distribution after 12 and 24 hours of treatment with 50 M free cisplatin. The loss of cells in G0/G1 and G2/M is attributable to the increase in the sub-G1 population. (Modified from Moreno et al., 2008.48 Used with permission.)

In addition to hydrophobic and hydrophilic drugs, amphiphilic anticancer drugs can also be entrapped in PLGA nanoparticles using either the nanoprecipitation method or the emulsification-solvent-diffusion methods. Cohen-Sela et al. entrapped an amphiphilic anticancer drug, mithramycin, in PLGA nanoparticles employing both the nanoprecipitation and emulsification-solvent-diffusion methods, and reported higher entrapment in the former (79.3 ± 3.1%) compared with the latter (40.8 ± 1.1%). The observed difference in drug entrapment could be attributed to the difference in the mechanisms of nanoparticles formation, as well as to the polarity of the solvent used in the two processes. In the emulsification-solvent-diffusion method, a partially watermiscible solvent was used, whereas for the nanoprecipitation method, the solvent was semipolar and water miscible. During the evaporation of the solvent in the emulsification-solvent-diffusion method, solvent diffuses through the aqueous phase, creating regions of local supersaturation near the interface, thus resulting in a lower entrapment efficiency.53 The diffusion method reported for PLGA nanoparticles has also been employed for the loading and delivery of dual therapeutic agents to enhance their efficacy and therapeutic potential. Loading of two drugs in PLGA nanoparticles is executable using the emulsion-diffusion method. As discussed earlier, formulation variables such as the molecular weight of PLGA, the nature of the surfactant, the ratio of the two


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drugs, the ratio of the organic phases, and the pH of the aqueous phase critically influence the drug-entrapment efficiency and the size of the particles and thus needs to be thoroughly optimized. With regard to dual drug entrapment, Song et al.54 formulated PLGA nanoparticles with simultaneous loading of one hydrophilic and one hydrophobic drug (vincristine sulfate and quercetin, respectively) using the oil/water emulsion solvent-evaporation method. The size of the nanoparticles loaded with the dual agents was around 139.5 nm, with a reasonable PDI. The entrapment efficiency of the nanoparticles loaded with vincristine and quercetin was found to be 92.84 % and 32.66 %, respectively.54 The same group also studied the simultaneous entrapment of two more hydrophilic, low-molecular-weight drugs in PLGA nanoparticles: vincristine sulfate and verapamil hydrochloride. They prepared the PLGA nanoparticles using oil/water emulsion-solvent evaporation and the salting-out method, and also studied 10 independent processing parameters to enhance the incorporation of the two drugs into PLGA nanoparticles. The particle size of the PLGA nanoparticles was obtained in the range of 111.4 nm, with a low PDI of 0.062. The entrapment efficiencies for vincristine and verapamil were determined to be 55.35% and 69.47%, respectively.55 Recently, Manchanda et al. reported the simultaneous incorporation of doxorubicin, a chemotherapeutic drug, and indocyanine green, a temperature-sensitive agent, into PLGA nanoparticles using the oil/water single-emulsion method. The different processing parameters were optimized for increasing the entrapment of two drugs, and the investigators proposed the applicability of dual drug-loaded PLGA nanoparticles for a combined hyperthermia and chemotherapy effect.56 In a different study, van Vlerken et al.57 constructed a paclitaxel-loaded polymer blend of a pH-responsive, rapid-releasing polymer poly(beta-amino ester) with a slowreleasing polymer PLGA-containing C6 ceramide, an apoptotic signaling molecule. These investigators studied the biodistribution and pharmacokinetic profile of paclitaxel and C6-ceramide in female nude mice bearing an orthotopic drug-sensitive MCF7 and a multi-drug-resistant MCF7 TR (MDR-1 positive) human breast adenocarcinoma after intravenously administering the polymer blend containing a high payload of bioactive. They found that the high concentration of paclitaxel in blood is due to longer retention time and superior tumor accretion relative to the free drug. They also found that the residence time of both agents at the tumor site was increased by PLGA/poly(beta-amino ester) blend nanoparticles. Overall, van Vlerken et al. concluded that PLGA, when incorporated in a polymeric blend, effectively controls the release and temporal release of loaded bioactive.57 A brief summary of reports for PLGA nanoparticles incorporated with anticancer drugs either alone or in combination is provided in Table 2. III.B. PEGylated PLGA Nanoparticles Despite the favorable biocompatibility and biodegradability of PLGA nanoparticles,58 a major problem encountered with the use of these colloidal carriers is that particles are amenable to rapid clearance from circulation by macrophages of the MPS immediately after their injection into the bloodstream. To circumvent this problem, nanoparticles



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formulated with the PLGA-PEG block co-polymer are currently being extensively investigated for drug delivery and relevant applications.59-61 Surface modification with PEG enables the nanoparticles to evade the MPS attack, with a concomitant increase in their plasma half-life, and in turn the area under the curve (AUC) concentration time.62 A hypothetical diagram illustrating the uptake of plain nanoparticles and PEGylated nanoparticles is shown in Figure 4. PEGylation of PLGA-based nanocarriers not only prolongs their time in the blood circulation, but also improves the drug payload, solubility, and kinetic stability while enhancing the targeting index and accessibility of the carrier toward the tumor site.63 For example, Danhier et al. developed paclitaxel-loaded, PEGylated, PLGA-based nanoparticles using the simple-emulsion method and the nanoprecipitation method, and showed higher entrapment efficiency for nanoparticles prepared via nanoprecipitation. As indicated by cell viability studies, paclitaxel-loaded nanoparticles are more cytotoxic toward human cervix carcinoma cells (HeLa), compared with free Taxol (half-maximal inhibitory concentration [IC50] 5.5 vs. 15.5 μg/mL). Interestingly, the same fraction of cells was found to be accumulated in the sub-G0 (apoptotic) phase upon exposure of HeLa cells to Taxol and paclitaxel-loaded nanoparticles. Paclitaxel-loaded nanoparticles also inhibited the in vivo tumor growth on transmissible lymphoid tumors more efficiently than Taxol.64 These results suggest that entrapment of paclitaxel within a PEGylated, PLGA-based nanocarrier alters its pharmacokinetics and biodistribution such that tumor-specific localization of the drug is significantly augmented, resulting in higher tumor-growth inhibition efficiency. In a similar approach, cisplatin-loaded PLGA-mPEG was prepared by Gryparis et 65 al. These investigators tested the dose tolerance of blank and cisplatin-loaded (2% w/w) PLGA-mPEG nanoparticles on BALB/c mice and assessed their in vivo anticancer activity on SCID mice xenografted with colorectal adenocarcinoma. They found that both blank and cisplatin-loaded, PEGylated nanoparticles could be administered in high doses (10 mg/kg) in normal BALB/c mice. HT 29 tumor-bearing SCID mice treated with cisplatin-loaded PLGA-mPEG nanoparticles showed higher survival rate and delayed tumor growth compared with animals treated with the free drug.65 In another study, in vitro degradation of the PLGA-mPEG nanoparticles in phosphate-buffered saline (pH 7.4) was found to increase the mPEG content (i.e., the mPEG:PLGA ratio) of the nanoparticles. As expected, PEGylation was found to prolong the circulation time of PLGA nanoparticles in BALB/c mice.38 Gryparis et al.65 further evaluated the in vitro cell uptake of cisplatin-loaded PLGAmPEG against the human prostate cancer LNCaP cell line, and observed that cell cytotoxicity was dependent on the ratio of PLGA/PEG in the PLGA-mPEG co-polymer employed for nanoparticle preparation. Cytotoxicity increased the ratio of PLGA/PEG in the polymer (Fig. 5). The nanoparticles with high PLGA/PEG ratio showed higher cell uptake. Further, cisplatin-loaded PLGA-mPEG nanoparticles were found to be equivalent to free cisplatin in terms of their cellular cytotoxicity (Fig. 6). As proposed by the authors, drug leakage during incubation with the cells was responsible for the comparable activity of drug-loaded nanoparticles and the free drug.66 In another study, Senthilkumar et al.67


Anticancer Drug Paclitaxel

Paclitaxel

Paclitaxel

Doxorubicin

In(III)-mesotetraphenylporphyrin Cisplatin

Etoposide

PLGA Nanoparticles/Surface Modification

Simple PLGA nanoparticles

PEGylated PLGA nanoparticles

PLGA nanoparticles conjugated with wheat germ agglutinin

cid-capped PLGA


Simple PLGA nanoparticles and PLGA microparticles

Simple PLGA and PLA nanoparticles

Double-emulsion solvent evaporation

Emulsification-evaporation

Nanoprecipitation

Solvent evaporation method with a release modifier isopropyl myristate

Simple emulsion and nanoprecipitation

High-pressure homogenization

Method of Preparation

TABLE 2. PLGA Nanoparticles for Anticancer Drug Delivery

Biodistribution and pharmacokinetics after radiolabeling with Tc-99m; both formulations showed higher radioactivity

Greater loading in microparticles but same entrapment efficiency; enhanced caspase-3 activity for microparticles and nanoparticles.

Much greater reduction in cell viability against LNCaP prostate tumor cells

155

48

154

52

44

Higher efficacy of wheat germ agglutinin-conjugated nanoparticles against the A549 and H1299 cell lines compared with Taxol Site-specific release; quick release at endolysosomal pH 4.0 but slow release at pH7.4; higher efficacy in MDA-MB-231 breast cancer cells

64

43

Reference

Higher entrapment by nanoprecipitation method, lower cell viability in MTT assay and higher tumor inhibition for paclitaxel-loaded nanoparticles compared with Taxol

Biphasic release profile; higher or comparable cytotoxicity against glioma C6 cells compared with Taxol

Purpose/ Outcome of Study

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Anticancer Drug Mithramycin

Vincristine sulfate and verapamil Vincristine sulfate and quercetin Paclitaxel and C6-ceramide





PLGA with rapid-releasing polymer poly (beta-amino ester)

TABLE 2. Continued


--

Oil/water emulsion solvent evaporation

Oil/water emulsion solvent evaporation method and salting-out process

Single- and doubleemulsion

Method of Preparation

Investigation of pharmacokinetic and biodistribution

57

54

55

Enhancement of drug-entrapment efficiencies

Optimization of loading of opposite nature of drugs in nanoparticles

53

Enhancement of encapsulation efficiency of hydrophilic agent; double-emulsion method yielded higher entrapment; inhibition of RAW264 macrophages by both solution and drug nanoparticles; ineffectiveness in the rat restenosis


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FIGURE 4. Hypothetical representation of uptake of plain and PEGylated PLGA nanoparticles.

FIGURE 5. Cytotoxicity of blank PLGA-mPEG nanoparticles on LNCaP cell. (Modified from Gryparis et al., 2007.66 Used with permission.)



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FIGURE 6. Cytotoxicity of free cisplatin and cisplatin-loaded PLGA-mPEG nanoparticles on LNCaP cells. (Modified from Gryparis et al., 2007.66 Used with permission.)

investigated the potential of PEGylated PLGA nanoparticles for the delivery of docetaxel to solid tumors. PEGylated, docetaxel-loaded PLGA nanoparticles enhanced the biological half-life of the drug while imparting considerable solid tumor accumulation.67 The success of PEGylated PLGA as a long-circulating drug carrier has prompted researchers to exploit this polymer for the targeted delivery of a wide range of anticancer drugs. Wong et al.68 encapsulated 5-fluorouracil in PLGA-PEG nanoparticles using a high-speed shearing double-emulsion method. The in vitro release of 5-fluorouracil showed a zero-order release from the second day. Chan et al.69 reported the development of a biodegradable core-shell nanoparticle system that exhibited the beneficial properties of liposomal and polymeric nanoparticles. The core-shell self-assembled nanoparticles were synthesized using the nanoprecipitation method, and consisted of a PLGA hydrophobic core, a soybean lecithin monolayer, and a PEG shell. The drug-release kinetics of docetaxel was found to be dependent on the amount of lipid coverage in the nanoparticles. Cytotoxicity was also investigated using an multi-target tracking assay using two different model human cell lines, HeLa and HepG2, and the biocompatibility of these particles in vitro was demonstrated. Finally, the authors concluded that the PLGA lecithin/PEG core-shell nanoparticles could be a useful new controlled release drug-delivery system for the delivery of anticancer bioactives. Although long-circulating nanocarriers formulated with PEGylated PLGA significantly increase the tumor localization of the payload, this strategy is not free from limitations. Capping of PLGA with PEG not only prevents the interaction between the nanocarriers and the opsonins, but also that between nanocarriers and the cell surface. For


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example, PEGylated liposomal doxorubicin was less efficient in entering the tumor cells and had a much lower AUCtumor/AUCplasma (0.31) compared with that of non-PEGylated liposomes (0.87). These reduced interactions inhibited the effective uptake of the payload by the tumor cells.70 We cannot rule out the possibility of similar problems in drug delivery using PLGA-PEG based nanocarriers. This necessitates the development of ligand-anchored PLGA nanocarriers that can actively target the tumor cells based on ligand-receptor binding interactions. An overall picture of recent developments in this area is given in the subsequent sections. III.C. Ligand-anchored PLGA Nanoparticles It has been already discussed that cancer cells, unlike normal cells, proliferate rapidly. One mechanism underlying this growth is the overexpression of receptors, which allows the uptake of growth factors via receptor-mediated endocytosis to proceed more efficiently than in normal cells. This mechanism could be used as a “Trojan horse” for site-specific delivery of anticancer agents, and is executable by decorating the surface of nanoparticles with antibodies or ligands that specifically bind to these receptors. This section reviews some representative examples of ligand-anchored PLGA nanocarriers and their application in site-specific cancer therapeutics. 1. Antibody-mediated Targeting Polymeric nanocarriers suffer from poor selectivity toward cancer cells. Therefore, targeting anticancer drugs specifically to the tumor site without affecting normal tissue remains a major hurdle for the scientific community. Localization of different PLGAbased drug carriers can be achieved by the conjugation of antibodies. The progression of cancer is usually associated with the overexpression of specific antigens (proteins) on cancer cells, which could serve as a tethering point for the antibody-anchored carriers. This quality can be exploited as an effective means of differentiating between cancerous and normal cells. With the goal of augmenting the selectivity of loaded anticancer bioactives, Kocbek et al.71 prepared antibody-functionalized PLGA nanoparticles for targeting breast cancer cells. Monoclonal antibodies (mAbs) recognizing the specific profile of the cytokeratins overexpressed on MCF-7 and MCF-10A neoT cells were attached to the fluorescein-loaded PLGA nanoparticles via covalent and noncovalent linking strategies. The occurrence and stability of antibodies on the nanoparticle surface were corroborated using a variety of complementary techniques, such as surface plasmon resonance, protein assay, flow cytometry, and fluorescence immunostaining. A preliminary test for the binding ability of formulated immunonanoparticles was carried out using MCF-7 or MCF-10A neo T-cell lysates. The nanoparticles with adsorbed mAbs were found to bind to the cell lysates, and the extent of binding was 3-fold higher than that of the control, uncoated nanoparticles. Conversely, Kocbek et al. found that covalently coupled mAb on nanoparticles resulted in a significant loss of binding affinity to cytokeratins in cell lysates. They also investigated the internalization of antibody-coupled nanoparticles by



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fluorescence microscopy and flow cytometry (Fig. 7). While both types of nanoparticles were found to be internalized inside the target MCF-10A neoT cells in monoculture, a different intracellular distribution pattern was observed when MCF-10A neoT cells were co-cultured with Caco-2 cells. Bare immunonanoparticles were localized solely to MCF-10A neo T-cells, whereas uncoated nanoparticles were distributed randomly (Fig. 8). Finally, these researchers determined the effectiveness and increased cellular internalization of antibody-conjugated PLGA nanoparticles and revealed the possibility of targeting of small or large bioactives to specific cells via antibody- conjugated PLGA nanoparticles.71 In a similar study, Kou et al. prepared paclitaxel-loaded PLGA nanoparticles coated with SM5-1 single-chain antibody (scFv) containing a polylysine (SMFvpoly-Lys) specific for a protein (230 kD) specifically expressed in hepatic carcinoma and breast cancer cells. Using a non-radioactive cell proliferation assay, they tested the cytotoxicity of paclitaxel loaded, scFv-coated nanoparticles against Ch-hep-3 cells, and demonstrated that the paclitaxel-loaded particles showed improved cytotoxicity over the nontargeted nanoparticle preparations. These studies suggested that cationic SMFvpoly-Lys could be generated and used as an effective targeting ligand for site-specific cancer therapeutics.72 In another study, Sun et al.73 formulated paclitaxel-loaded, PLGA/ montmorillonite (PLGA-MMT) nanoparticles, which were further decorated with the

FIGURE 7. (A) Internalization of fluorescein-loaded PLGA nanoparticles coated with mAb and (B) uncoated nanoparticles into MCF-10A neoT cells after 12 hours of incubation. A perinuclear localization of internalized nanoparticles (green) was observed in both cases. (Modified from Kocbek et al., 2007.71 Used with permission.).

FIGURE 8. Fluorescence microscope image of a co-culture of MCF-10A neoT and Caco-2 cells incubated with fluorescein-loaded PLGA nanoparticles coated with mAb (A) and non-coated nanoparticles (B) after 24 hours of incubation. Immunonanoparticles (green) only entered MCF10A neoT cells, while non-coated nanoparticles entered both types of cells. The cells stained blue are Caco-2 cells. (Modified from Kocbek et al., 2007.71 Used with permission.).


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human epidermal growth factor receptor-2 (HER2) antibody trastuzumab for selective targeting and killing of HER2-overexpressing breast cancer cells. They determined the cellular uptake of nanoparticles after 2 hours of incubation by using coumarin-6 dye in Caco-2 cells and SK-BR-3 cells (Fig. 9), and found that trastuzumab decoration significantly improved the cellular uptake either by Caco-2 cells or by SK-BR-3 cells by 1.24-fold compared with the non-targeted control. The researchers also evaluated the in vitro cytotoxicity of these prepared particles and found that the surface conjugation with antibody increased the therapeutic effects of the drug formulated in the nanoparticles by 12.74 and 13.11 times compared with bare nanoparticles and free Taxol. In another study, Gao et al. formulated PE38KDEL-I-loaded PLGA nanoparticles conjugated with F (ab’) fragments of a humanized SM5-1 monoclonal antibody (PENP-S). These antibody-conjugated nanoparticles showed high intracellular uptake against the hepatocellular carcinoma (HCC) cell lines through the interaction between SM5-1 antibody conjugated on the surface of nanoparticles and its corresponding antigen, which is specifically expressed on the surface of HCC cells. The antibody-conjugated nanoparticles showed significantly reduced tumor burden in a SM5-1 binding protein, overexpressing tumor xenograft model. An approximately 83% reduction in tumor burden was observed in 0.5 mg/kg PE-NP-S-treated mice versus either 0% for all other control groups or 33% (2 of 6) for 0.5 mg/kg Mut-I-treated group. They also reported a 4-fold higher median lethal dose and lesser immunogenicity for PE-NP-S than for Mut-I. The positive attributes of the study clearly establish the potential of these antibody-anchored nanoparticles in targeted cancer therapy.74 A comprehensive overview of different antibodies anchored or conjugated to PLGA nanoparticles is provided in Table 3. Despite their widespread applicability, one major problem associated with the use of antibodies as targeting agents is their large size and inherent immunogenicity, which can cause the conjugated nanoparticles to diffuse poorly through the biological barriers. This problem, however, has encouraged researchers to explore the efficacy of other affinity ligands such as short peptides and small molecules for tumor-specific drug targeting.

FIGURE 9. Confocal laser scanning microscopy of SK-BR-3 breast cancer cells after 1 hour of incubation with the courmine-6 loaded (a) PLGA/ MMT NPs or (b) PLGA/MMT-HER nanoparticles at 0.125 mg/ml nanoparticles concentration at 37°C. (Modified from Sun et al., 2008.73 Used with permission.).



Monoclonal antibody

SM5-1 single-chain antibody (scFv) containing polylysine Trastuzumab

F (ab’) fragments of a humanized SM5-1 monoclonal antibody (PE-NP-S) Humanized anti-HER2 monoclonal antibody (rhuMAbHER2)

Peripheral antibodies

Antibodies for EGFR

PLGA nanoparticles

Paclitaxel-loaded PLGA nanoparticles

Paclitaxel-loaded PLGA/ MMT nanoparticles

PLGA nanoparticles

PE38KDEL (model protein)-loaded PLGA nanoparticles

PLGA nanoparticles loaded with camptothecin

PLGA nanoparticles loaded with rapamycin

EGFR, epidermal growth factor receptor

Antibody/Source of Antibody

PLGA Nanoparticles/ Surface Modification

EGFR expressed on breast cancer cells

Fas receptor (CD95/ Apo-1) protein

HER2 expressed on breast cancer cell lines

SM5-1-binding protein expressed on hepatocellular carcinoma cell lines

Breast cancer cells

Protein of MW 230Da, specifically expressed in hepatic carcinoma and breast cancer cells

Cytokeratins expressed on MCF-7 and MCF10A neoT cells

Target Protein/ Receptor

TABLE 3 . Antibody-coated PLGA Nanoparticles for Cancer Therapeutics

Superior antiproliferative activity compared with free drug

Effective internalization in HCT116 cells; IC50 of 0.37 ng/mL for antibody functionalized nanoparticles compared with camptothecin solution (21.8 ng/mL)

Superior in vitro cytotoxicity in HER2 cells; higher antitumor activity in HER2-overexpressing tumor-bearing tumor model; reduced systemic toxicity as revealed by reduction in maximum tolerated dose (2.92 vs. 0.92 mg/kg)

Significant reduction in tumor burden in SM5-1-binding protein-overexpressing tumor xenograft model

Improved cellular uptake in SK-BR-3 cells by 1.24 fold; 12.74 times higher therapeutic effect than bare nanoparticles and 13.11 times higher than Taxol

Improved cellular cytotoxicity against Chhep-3 cells

Targeting of breast tumor cells; increased cellular internalization


158

157

156

74

73

72

71

Reference


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2. Peptide-mediated Targeting The growth of tumor tissue may be related to its ability to generate adequate blood supply both inside the tumor mass and in its vicinity. Cancer therapeutics based on targeting the progression of tumor vasculature has therefore gained much research interest. The metastatic capability of a tumor may be regulated by targeting the tumor vasculature.75 The formation of new blood vessels, or angiogenesis, is characterized by a cascade of events that include the invasion, migration, and proliferation of smooth muscle and endothelial cells, followed by the degradation of the basement membrane and the formation of new luminal structures.8 Impairing the targeting ability of tumor vasculature has emerged as an attractive strategy for the tumor-specific delivery of anticancer agents and relevant bioactives for the following reasons:76: i) specificity in targeting can be achieved due to the expression of proliferative tumor-derived endothelial cells; ii) angiogenesis in the adult is limited to wound healing, ovulation, pregnancy, and atherosclerosis, so this method causes minimal toxicity; iii) the method enhances and improves the accessibility of drugs compared with other drugs that are required to penetrate inside the tumor mass; iv) angiogenesis-mediated cancer therapeutics is independent of tumor cell resistance mechanisms because of low mutations within the vasculature; and v) the strategy has broad applications in terms of therapeutics, because the growth and thus the survival of tumor cells are dependent upon angiogenesis. A variety of pro-angiogenic factors are infiltrated by tumor and host cells including platelet-derived growth factor, fibroblast growth factor, vascular endothelial growth factor, angiogenin, interleukin-8 (IL-8), angiotropin, platelet-derived endothelial cell growth factor, transforming growth factor-α and -β, epidermal growth factor, and tumor necrosis factor-α.77 A brief summary of the various anti-angiogenic agents employed for site-specific cancer therapeutics is provided in Table 4. Targeting tumor angiogenesis using nanotechnology is advantageous because it holds the possibility of overcoming physiological barriers that exclude the diffusion of nanoparticles through the tumor, irrespective of tumor type. However, the most prevalent drawback associated with the delivery of angiogenesis inhibitors is their instability in the biological environment and their high dosage requirements. To address these disadvantages, various nanocarrier approaches have been tried by different researchers. A variety of surface targets have been investigated for the impairment of angiogenesis in tumor tissue; these include vascular endothelial growth factor receptors, αvβ3 integrins, matrix metalloproteinase receptors, and vascular cell adhesion molecule-1.8 A thorough discussion of these targets is beyond the scope of the present review, but they are discussed briefly in Table 5. In addition to the methods described above, PLGA nanoparticles can also be surface engineered with different target moieties specific for tumor vasculature. There have been very few studies in which PLGA nanoparticles have been surface engineered for targeting the angiogenesis pathways of tumors. Danhier et al.78 formulated PEGylated PLGA-based nanoparticles grafted with the Arg-Gly-Asp (RGD) or the RGD-peptidomimetic (RGDp) peptide for enhancing the targeting index and antitumor efficacy of



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TABLE 4. Antiangiogenic Drugs and Their Molecular Targets76 Anti-angiogenic Drug

Target

Mechanism of Action

Trastuzumab (Herceptin)

Growth factor receptors

Inhibition of TGF-β, PAI-1, angiopoietin, and VEGF production, and stimulation of TSP-1 production

ZD1839 (Tarceva)


Inhibition of VEGF, bFGF, TGF-α, and IL-8 production Inhibition of Bcr-Abl kinase-, PDGF receptor-, and KIT-mediated effects

(Iressa),

erlotinib

Growth factor receptors Imatinib mesylate (Gleevec) Interferon-α


Inhibition of bFGF production

Bevacizumab (Avastin) (humanized antibody), VEGF-trap (decoy VEGF receptor)

Vascular cells (endothelial cells and/or pericytes)

Inhibition of the effects of VEGF

Endostatin (α5β1 integrin ligand), angiostatin (αvβ3 integrin and ATP synthase ligand), tumstatin (αvβ3 ligand), arrestin (α1β1 integrin ligand), LM609 (Vitaxin) (antibody against αvβ3)

Endothelial cell function

Inhibition of endothelial cell proliferation, migration, and survival

Celecoxib (Celebrex), rofecoxib (Vioxx)


Inhibition of COX-2 and its downstream mediator PGE2, which promotes endothelial cell proliferation and survival

AE941 (Neovastat) (shark cartilage)


Inhibition of VEGF; stimulation of tPA

PDGF, platelet-derived growth factor; bFGF, basic fibroblast growth factor; VEGF, vascular endothelial growth factor; TGF, transforming growth factor; KIT

paclitaxel. As expected, RGD-grafted nanoparticles showed higher intracellular uptake in human umbilical vein endothelial cells than their non-targeted (peptide non-conjugated) counterparts through the binding interaction between RGD immobilized on the surface of PLGA-PEG and αvβ3 integrin overexpressed on the malignant cell surface. Using that same rationale, RGD-conjugated, PEGylated PLGA nanoparticles have been used to improve the tumor-specific accumulation and antitumor efficacy of doxorubicin. Surface modification of PLGA with PEG and RGD peptide led to an enhanced loading efficiency, which also alleviated the burst release exhibited by the plain nanoparticles. Subsequently, the RGD-targeted nanoparticles showed high binding affinity toward the integrin-expressing malignant cells compared with the non-integrin-expressing cells. The nanoparticles also significantly inhibited tumor growth in malignant cancer cells.79 Luo et al.80 investigated the activity of peptide-conjugated PEGylated PLGA nanoparticles for targeted delivery to lymphatic metastatic tumors. Nanoparticles of maleimide-PEG-PLGA were targeted with LyP-1 peptide (H-Cys1-Cys2-Gly3-Asn4Lys5-Arg6-Thr7-Arg8-Gly9-Cys10-OH) through the conjugation of the sulfhydryl group of Lyp-1 to the maleimide located at the distal end of PEG on the nanoparticle surface.


I-Sc-7269-DMN showed significant tumor inhibition compared with other treatment (131I control, 131I-Sc-7269 radiolabeled antibody, 131 I-labeled DMN)

DMNs conjugated to radiolabeled 131Ianti-VEGF mAbs

Extensive co-localization of immune liposomes coupled with the anti-VCAM-1 monoclonal antibody in endothelial cells of Colo 677 xenograft tumors of mice

30-fold enhancement in micelle internalization with 76% RGD-functionalized doxorubicin-loaded micelles compared with the nontargeted micelles

Doxorubicin-loaded micelles coupled with differential RGD peptide coupling (based on PEG chains) VCAM-1 targeted immunoliposomes

Receptor binding studies of RGD mimetic showed the lesser IC50 values (0.04 μM for purified αvβ3) compared with alone αvβ3 (5.5 μM for αvβ5)

RGD-coupled nanoparticles

164

163

162

161

160

159

Reference

VEGF, vascular endothelial growth factor; VCAM-1, vascular cell adhesion molecule-1; DMN, dextran magnetic nanoparticle

VCAM-1

αvβ3 Integrin (arginine-glycineaspartic acid, or RGD)

Formulation showed better efficacy compared with anti-VEGF-2 mAb alone or conventional radioimmunotherapy

Radioisotope-labeled polymerized liposome (anti-VEGFR-2 mAb-labeled liposome labeled with 90Y) 131

Conjugation of recombinant VEGF121 molecule on nanoparticles surface; selective binding of dendrimer to VEGFR-2 and internalization by endocytic pathways

Boronated dendrimers

VEGF and VEGFR-2

Comments

Nanocarrier System

Target

TABLE 5. Surface-engineered Nanocarrier Systems for Angiogenesis-mediated Tumor Targeting

22 A.K. Jain et al.



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The researchers tested the efficacy of peptide-conjugated PEGylated PLGA nanoparticles against the BxPC-3 human pancreatic cancer cell line, and found that the cellular uptake of LyP-1 nanoparticles was approximately four times than that of PEG-PLGA nanoparticles without LyP-1 (Fig. 10). The in vivo trafficking of the nanoparticles were also investigated in nude BALB/c nu/nu mice, and an 8-fold higher uptake of LyP-1 nanoparticles was found in metastasized lymph nodes compared with plain nanoparticles (Fig. 11).80 3. Folate-linked PLGA Nanoparticles for Cancer Therapeutics Functionalization of nanoparticles with small molecules as targeting agents has recently been described as an alternative targeting strategy with the potential for major biomedical advances. In this particular context, tumor-specific drug delivery using folic acid as a targeting ligand deserves special mention because folate receptors are ubiquitously overexpressed on a wide variety of cancer cells, including lung, colon,81 ovarian,82,83 kidney, breast, and choroid plexus brain tumors.84 Further, the use of the folic acid as a targeting ligand offers unique advantages such as favorable pharmacokinetic properties of folic acid conjugates due to the small size of the ligand; a reduced likelihood of immunogenicity, which allows the repeated administration of the nanocarriers; low cost and availability; simple and defined conjugation chemistry; high receptor affinity; and lack of normal tissue receptor expression.85 Because of these positive attributes of

FIGURE 10. Binding capacity of fluorescein isothiocyanate (FITC)-labeled LyP-1 to BxPC-3 cells. No obvious FITC fluorescence was detected by microscopy when cells were incubated with 1 mg/ mL FITC (A1 and A2), and nearly all cells were stained with green fluorescence when incubated with 10 μmol/L FITC-labeled LyP-1 (B1 and B2). The FITC-positive cells were quantified by flow cytometry at different concentrations of FITC-labeled LyP-1. (Modified from Luo et al., 2009.80 Used with permission.)


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FIGURE 11. LyP-1-nanoparticles’ binding affinity to metastasis lymph nodes. (A1) Nude BALB/c nu/nu mice were injected into the nail pad with 1×106 BxPC-3 cells to induce lymphatic metastasis models for 3 weeks (A2) Mice received 6-coumarin loaded nanoparticles (A3) 6-coumarin loaded LyP-1-nanoparticles, via the nail pad at a dose of 60 mg/kg body weight. (Modified from Luo et al., 2009.80 Used with permission.)

folate receptor-mediated targeting, folic acid has been conjugated with a wide variety of drug-loaded nanocarriers, including dendrimers,86,87 polymeric micelles,88 liposomes,89,90 nanoparticles,91 and magnetic resonance imaging agents92,93 to direct them selectively to the tumor site. Functionalization of nanocarriers with folic acid makes it possible to distinguish between malignant and normal cells, and facilitates their internalization inside the cancer cells through receptor-mediated endocytosis (Fig. 12).94 For these same reasons, folic acid has been tethered onto the surface of PLGA-based nanocarriers for augmenting their tumor-specific localization, and consequently, their antitumor activity. The use of folic acid for the active targeting of PLGA nanoparticles to a great extent was due to the work of Kim et al.95 These investigators coated anionic PLGA nanoparticles with cationic di-block co-polymer, poly(L-lysine)-PEG-folate

FIGURE 12. Schematic representation of receptor-mediated endocytosis.



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(PLL-PEG-FOL) for enhancing the selectivity to the cancer cells. By employing the flow-cytometry method and confocal image analysis, the extent of cellular uptake was evaluated in KB and A549 cells in the presence and absence of excess free folate (Fig. 13). PLL-PEG-FOL-coated PLGA nanoparticles demonstrated a far greater extent of cellular uptake to KB cells, suggesting that they were mainly taken up by folate receptor-mediated endocytosis. The positive outcome of this study suggests that PLL-PEGFOL-coated PLGA nanoparticles could be potentially applied for cancer cell-targeted delivery of various therapeutic agents.95 Zhang et al.96 prepared doxorubicin-loaded nanoparticles of two different conjugates, vitamin E-TPGS-folate and doxorubicin-poly(lactide-co-glycolide)-vitamin ETPGS, using the solvent extraction/evaporation method. They investigated the cellular uptake of the prepared nanoparticles and found that cellular uptake of nanoparticles was dependent on content of targeting the TPGS-folate conjugate in the nanoparticles. After 30 minutes of incubation, MCF-7 and C6 cells cultured with nanoparticles con-

FIGURE 13. Cellular uptake of PLGA nanoparticles under various conditions. (A) KB cells (folate receptor positive carcinoma) were treated with plain PLGA nanoparticles (black line) or PLL-PEGFOL coated PLGA nanoparticles (blue line), or without the nanoparticles (red line) in serum-free media containing physiological levels of free folate. (B) A549 cells (folate receptor negative carcinoma) incubated in media containing non-coated PLGA nanoparticles or PLL-PEG-FOL-coated PLGA nanoparticles. (C) The effect of free folate on the cellular uptake of PLGA nanoparticles coated with or without PLL-PEGFOL for KB cells. Cells were incubated in the media with or without 1 mM free folate. (D) The effect of serum proteins in the media on the cellular uptake of PLGA nanoparticles for KB cells. Cells were treated with non-coated PLGA nanoparticles or PLL-PEGFOL-coated PLGA nanoparticles in the media with or without 10% fetal bovine serum. Control represents the fluorescence emitted from naked KB cells. (Modified from Kim et al., 2005.95 Used with permission.)


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taining 50% TPGS-folate showed 1.5 and 1.7 times higher uptake than cells exposed to nanoparticles deprived of the TPGS-folate component. They also reported a reduction in the IC50 of doxorubicin after 24 hours of incubation. In a similar study, Esmaeili et al. prepared folate-conjugated, PEGylated PLGA nanoparticles for the delivery of docetaxel. They synthesized a folate-conjugated di-block co-polymer by coupling the PLGA-PEG-NH2 di-block co-polymer with activated folic acid. Using an emulsification/solvent diffusion method, they prepared docetaxel-loaded PLGA nanoparticles with an average size of 200 nm. As observed in the other studies, the folate-targeted nanoparticles showed a greater extent of intracellular uptake in folate receptor-positive cancer cells (SKOV3) compared with the the non-targeted controls. These results suggested that folate-targeted docetaxel nanoparticles could be a potentially useful delivery system for targeting folate receptor-overexpressed cancer cells.97 Ebrahimnejad et al. designed a cancer-specific PLGA nanoparticulate delivery system for the effective delivery of a highly hydrophobic anticancer metabolite of irinotecan, SN-38 (7-ethyl-10-hydroxycamptothecin). They prepared SN-38 nanoparticles using the PLGA-PEG-FOL conjugate in the emulsification/solvent-evaporation method, and found that SN-38-targeted nanoparticles showed a greater cytotoxicity toward HT-29 cancer cells than SN-38-nontargeted nanoparticles.85 4. Aptamer-Functionalized PLGA Nanoparticles for Cancer Therapeutics Aptamers are the molecular probes that bind specifically to the biomarkers expressed in cancer cells.98 DNA aptamers can be employed for the diagnosis and selective targeting of cancer cells. DNA aptamers are created by a specific technique called the cell-based systemic evolution of ligands by exponential enrichment (SELEX) process. Aptamers employ whole diseased cells as their target. The fundamental challenge in cancer treatment lies in the selective molecular recognition of disease-specific biomarkers such as proteins between normal and cancer cells. Specific types of proteins are overexpressed in cancer cells due to genetic abnormalities such as mutations, translocations, or fusion events.99 Basically the aptamers are group of selected amplified sequences that form a random library of single-stranded DNA or RNA with high affinity toward target molecules such as proteins and drugs.100 Cell-SELEX aptamers have the unique capability of binding specifically to the whole cell, which makes them ideal candidates for detecting occult tumor cells in different types of samples. Because bodily fluids contain very minute quantities of cancer cells, tumor cells should be enriched before detection. Aptamer-conjugated magneto-fluorescent nanoparticles have been effectively employed in tumor cell enrichment.98 Because of their non-immunogenic properties and stability over a broad range of pH (4–9), temperatures, and organic solvents, aptamers have shown considerable promise in the active targeting of tumors.101,102 Encouraged by the positive aspects of an aptamer-based targeting strategy, Farokhzad et al.103 formulated aptamer-decorated PLGA nanoparticles for tumor-specific delivery of the anticancer drug docetaxel. They synthesized nanoparticles of docetaxel-loaded PLGA-block-PEG co-polymer and functionalized their surface with A10 2’-fluoropy-



27

rimidine RNA aptamers specific for prostate-specific membrane antigen (PSMA). Cytotoxicity studies were conducted in LNCaP cells expressing the PSMA protein (Fig. 14). Their results corroborated that aptamer-conjugated, docetaxel-loaded PLGA-PEG nanoparticles were significantly more cytotoxic than control nanoparticles deprived of aptamers. Further, these aptamer-conjugated nanoparticles showed complete tumor reduction in five out of seven LNCaP xenograft nude mice, along with a 100% survival rate in 109 days, while their non-targeted control (i.e., docetaxel-loaded nanoparticles devoid of aptamers) showed complete tumor reduction but only 57% survival rates (docetaxel was associated with only 14% cell survival). Similarly, Dhar et al.104 developed cisplatin-loaded, PEGylated PLGA nanoparticles specifically targeted to prostate cells by conjugating them with PSMA-targeting aptamers. These engineered PLGA nanoparticles were loaded with lethal dose of platinum (IV) compound c, t, c-[Pt(NH3)2(O2CCH2CH2 CH2CH2CH3)2Cl2]. As evidenced by fluorescence microscopy, these aptamer-engineered Pt IV-encapsulated nanoparticles were internalized into PSMA-specific LNCaP cells by receptor-mediated endocytosis (Fig. 15). 5. Miscellaneous Targeting Ligands Other than the targeting agents discussed in the preceding sections, a variety of other targeting ligands such as transferrin and hyaluronate have also been evaluated for the targeted delivery of PLGA-based nanocarriers. For example, Sahoo and Labhasetwar105 studied the efficiency of transferrin-coupled PLGA nanoparticles for the targeted delivery of paclitaxel in cancer cells. Compared with unconjugated nanoparticles or drug solutions, transferrin-conjugated nanoparticles showed increased activity in MCF-7 and

FIGURE 14. MTT assay to determine the differential cytotoxicity of Dtxl-encapsulated nanoparticle-Apt bioconjugates (Dtxl-NP-Apt), Dtxl-encapsulated nanoparticles lacking the A10 PSMA Apt (Dtxl-NP), control NP-Apt bioconjugates without Dtxl (NP-Apt), and control nanoparticles without Dtxl (NP) after incubation with LNCaP prostate epithelial cells. Nanoparticles were incubated with cells for 30 minutes (left) or 2 hours (right), and the cells were subsequently washed and incubated in media for a total of 72 hours before assessing cell viability in each group. (Modified from Farokhzad et al., 2006.103 Used with permission.)


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FIGURE 15. Detection of endosome formation and cellular uptake of Pt-NP-Apt in LNCaP cells by fluorescence microscopy. Green fluorescent 22-NBD-cholesterol and platinum (IV) compound were encapsulated in the PLGA-b-PEG nanoparticles, and PSMA aptamers were conjugated to the surface of the particles. The early endosomes were visualized in red by using the early endosomes marker EEA-1. (Modified from Dhar et al., 2008.104 Used with permission.).

MCF-7/Adr cells. This increased activity was attributed to the greater cellular uptake and reduced exocytosis compared with the unconjugated nanoparticles, which enhanced the intracellular paclitaxel level. The researchers suggested that by sustaining the intracellular drug retention, drug resistance could be circumvented by the concomitant increase in antiproliferative activity. Finally, they concluded that transferrin-conjugated nanoparticles were taken up by cells via the transferrin receptors, as opposed to the unconjugated nanoparticles, which were taken up by normal endocytic pathways (Fig. 16).

FIGURE 16. Cellular uptake and exocytosis of Tf-conjugated and unconjugated nanoparticles. (A) Uptake of Tf-conjugated nanoparticles (NPs-Tf) and unconjugated nanoparticles (NPs) in MCF-7 cells. (B) Exocytosis of Tf-conjugated and unconjugated nanoparticles in MCF-7 cells. (Modified from Sahoo and Labhasetwar, 2005.105 Used with permission.)



29

In another study, Zheng et al.106 reported the formulation of transferrin conjugated lipid-coated PLGA nanoparticles, containing an aromatase inhibitor. SKBR-3 breast cancer cells model was employed for the evaluation of the aromatase inhibition activity. Compared with the plane nanoparticles, the ligand coupled nanoparticles presented lower IC50 value. Finally, the researchers concluded that aromatase inhibition activity was enhanced for the transferrin coupled nanoparticles due to their selective uptake by cancer cells. Another targeting agent which deserves special mention in this regard is hyaluronic acid, an anionic, non-sulfated aminoglycan, the activated receptors of which are overexpressed by CD44 and RHAMM (receptor for hyaluronan-mediated motility) on tumor cells, which is lacking in their non-tumorigenic counterparts. Attracted by the potential of hyaluronic acid-mediated targeting, Yadav et al.107 developed doxorubicinloaded, PEGylated PLGA nanoparticles and compared their activity with hyaluronic acid non-conjugated, doxorubicin-loaded, non-PEGylated and monomethoxy-PEGconjugated PLGA nanoparticles. The authors observed an increased targeting potential of the drug by ligand-conjugated, PEGylated PLGA nanoparticles compared with the PEGylated-only nanoparticulate system. These results suggest that tumor-specific localization of PEGylated PLGA nanoparticles was considerably improved by conjugating them with the targeting ligand, which in turn synergizes their tumor-growth inhibitory efficiency over the PEGylated-only PLGA nanoparticulate system.107 More recently, Lee et al.108 synthesized PLGA-grafted hyaluronic acid co-polymers and utilized them as target-specific micelle carriers for doxorubicin. In vitro studies against hyaluronic acidoverexpressed HCT-116 cells revealed that doxorubicin-loaded, hyaluronic acid-grafted PLGA micelle nanoparticles exhibited higher cellular uptake and greater cytotoxicity than free drug due to their selective uptake by the target cells via receptor-mediated endocytosis.108 6. Ligand-anchored PLGA Nanocarriers: A Critical Evaluation Although extensive research has focused on ligand-decorated PLGA nanocarriers, it is worthy of mention that these engineered nanocarriers also suffer from limitations. First, the targeting molecules can expose the nanocarriers to the RES during the circulation. Although in vitro experiments have demonstrated a high accumulation of these nanocarriers into the target cells, a number of in vivo studies have revealed that a significant percentage of the injected dose per gram accumulates in the RES organs (liver or spleen). This is undesirable unless these organs are the intended targets, because anticancer drugs may damage the MPS organs and/or be destroyed before reaching the tumor site.107 Second, while the long circulation time is crucial for selective distribution of the nanocarriers at the tumor sites, the recognition of these targeted nanocarriers by the MPS expedites their clearance during circulation. For example, the addition of antibodies on the surface of PLGA-PEG nanoparticles may conciliate the shielding effect of the PEG chains associated with PLGA core. These limitations partially account for the lack of actively targeted nanocarriers on the market. Based on similar considerations, PLGA nanocarriers targeted with small molecules such as folic acid seems to be a bet-


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ter option compared with their antibody- or peptide-targeted variants. Because of their small size compared with peptides and antibodies, nanoparticles targeted with folate or other small molecules provide the added advantage of functionalizing the nanoparticle surface with an increased number of ligands and limited steric constraints. However, despite these limitations, the introduction of targeting ligands onto the surface of PLGA nanoparticles improves the anticancer effect of the entrapped drug by facilitating the cellular uptake and intracellular retention of the drug carriers, which in turn augments their antitumor efficacy. IV. GENE DELIVERY BY PLGA NANOPARTICLES: A CELLULAR APPROACH TO CANCER TREATMENT Gene delivery is considered the most promising tool for targeted drug delivery. This technique can be utilized for treating a variety of infectious diseases such as monogenic diseases and cancer.109 The transfer of a gene of interest into a diseased cell is the basic core of gene therapy. Gene delivery can be executed through viral and non-viral vectors. Viral vectors include recombinant retrovirus, adenovirus, or adeno-associated virus,110-113 and non-viral vectors include cationic polymers,114,115 chylomicron remnants,116 particle bombardment,117 gene guns,118 electroporation,119 and liposomes.120 A gene or any macromolecule on its way from outside of the cells to the cellular milieu meets numerous hurdles, such as poor permeability, membrane non-selectivity, and degradation in the endo-lysosomal environment. A variety of non-viral novel carriers have been developed for intracellular gene delivery specifically for cancer therapeutics.121,122 PLGA nanoparticles have also been explored as a delivery vehicle for DNA, so that the target gene is effortlessly transfected with the cancer cells. For this purpose, the surface properties of PLGA nanoparticles such as surface coating or charge have to be appropriately tailored to make them an efficient carrier for DNA transfer into cancer cells. For example, Prabha and Labhasetwar123 determined the antiproliferative activity of wild-type (wt) p53 geneloaded PLGA nanoparticles prepared using the multiple-emulsion-solvent-evaporation technique. Compared with naked wt-p53 DNA or wt-p53 DNA complexed with a commercially available transfecting agent (Lipofectamine; Invitrogen, Carlsbad, CA), wt-p53 DNA-loaded nanoparticles exhibited significantly higher anti-proliferative activity in a sustained manner. Similar results were obtained for p53 mRNA levels. The authors also observed a higher intracellular localization of fluorescently labeled DNA-PLGA nanoparticles in a breast cancer cell line compared with the fluorescently labeled DNA alone.123 In another study, He et al.124 prepared recombinant plasmid P (EGFP-AFP) encapsulated PLGA nanoparticles (mean entrapment efficiency 91.25%) using the double-emulsion evaporation technique. A total of 80.14% DNA was found to be localized in the liver after 1-hour infusion of (32)P-DNA-PLGA nanoparticles into the caudal vein of mice. The expression of DNA encapsulated in nanoparticles was much higher than that in naked DNA, and human hepatocellular carcinoma SMMC-7721 cells showed higher sensitivity toward ganciclovir than human normal parenchymal Chang liver cells. Similar studies were performed by Koby et al.,125 who addressed different formulation



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considerations such as sonication time and the use of minimal additives to minimize the toxicity issues associated with PLGA nanoparticles. The researchers formulated DNA-loaded PLGA nanoparticles using the same method but with lower entrapment efficiency (80%). Endolysomal localization of DNA-loaded PLGA nanoparticles was detected when tested against different cell lines. No change in cellular viability was observed. In vitro studies against COS-7 and Cf2th cell lines revealed a 250-fold higher transfection efficiency for DNA-loaded nanoparticles compared with native DNA (Fig. 17). Transfection efficiency was further improved by coating PLGA nanoparticles with a cationic polymer such as chitosan.126 Because they have positive charges (13.5–60.4 mV), chitosan-coated nanoparticles could be easily loaded with negatively charged antisense oligonucleotide OMR with a phosphorothioate backbone (5′-2′-O-methyl [C(ps) A(ps)GUUAGGGUU (ps)A(ps)G]-3′). As evident from confocal laser microscopy studies, the chitosan/PLGA nanoparticles loaded with antisense oligonucleotide were efficiently taken up by A549 cells. Recently, Díez et al.127 developed a novel cationic, PLGA-based nanoparticulate system that was imparted a positive charge by modification with 1,2-dioleoyl-3-(trimethyl ammonium) propane and targeted with asialofetuin. Positively charged nanoparticles loaded with luciferase and the IL-2 gene showed enhanced transfection efficiency in HeLa cells (which are defective in asialoglycoprotein receptors), compared with their non-targeted counterparts and blank DNA. A similar cationic vector was used by the same group109 for the delivery of the luciferase gene and its subsequent expression in the hepatic cells after intravenous administration. IL-12 was either encapsulated or adsorbed on the nanoparticulate surface. A 5- and 12-fold higher transfection activity in the liver was observed compared with non-targeted (plain) complexes or naked pCMV DNA, respectively. Following the administration of IL-12-encapsulated nanoparticles, an approximately 75% reduction in tumor mass was observed in BNL, a tumor-bearing animal, whereas no tumor-growth inhibitory effect was observed in naked pCMV DNA.

FIGURE 17. (A) Intracellular trafficking of TOTO-3 labeled oligonucleotides in MDA-kb2 cells. (B) Cells were treated with TOTO-3 labeled oligos in the presence of DharmaFECT (Thermo Scientific, Lafayette, CO) encapsulated in nanoparticles. Cells were imaged at the end of day 3 after counterstaining for lysosomes using LysoTracker Green (Invitrogen). Images obtained using rhodamine and fluorescein filters were overlaid to determine the oligonucleotide localization. (Modified from Koby et al., 2007.125 Used with permission.)


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These engineered nanoparticles were also found to enhance the level of IL-12 and IFN-γ in the serum, which was crucial for the complete tumor reduction.. A number of studies have been carried out for the delivery of DNA or a specific gene via PLGA nanoparticles and the results are summarized in Table 6. V. SIRNA DELIVERY BY PLGA NANOPARTICLES: A CELLULAR APPROACH TO CANCER TREATMENT Gene silencing via short interfering RNA (siRNA) is currently the fastest growing sector of the antigene field for target validation and therapeutic applications. However, systematic deliverability of siRNA into target cells is often challenged by their rapid degradation128 and poor cell-penetration properties.129 To circumvent these problems, a broad spectrum of viral and non-viral vectors have been successfully utilized to improve the targeted delivery of siRNA to cancer cells and protect them from premature degradation in the biological milieu. PLGA-based systems have also been investigated for the targeted delivery of siRNA to cancer cells and to induce gene silencing. Nguyen et al.130 employed a PLGA-based nanosystem formulated with biodegradable branched polyesters DEAPA-(68)-PVA-PLGA, a derivative of poly(vinyl 3-(dialkylamino)alkyl carbamate-co-vinylacetate-co-vinylalcohol)-graft-poly-(D,L-lactide-co-glycolide)-containing diethylaminopropylamine (DEAPA) as a delivery vehicle for siRNA (anti-luciferase siRNA: 5′-GAUUAUGUCCGGUUAUGUA-3′). siRNA release studies were performed and correlated to the nanoparticle degradation. In vitro knockdown assay was used to assess the potential of the nanoparticles as siRNA carriers in a human lung epithelial cell line, H1299 luc. These nanoparticles achieved 80% to 90% knockdown of the luciferase reporter gene, with only 5 pmol anti-luc siRNA, even after nebulization. The authors concluded that amine-modified-PVA–PLGA/siRNA nanoparticles could be a promising siRNA carrier for pulmonary gene delivery due to their fast degradation and potent gene knockdown profile.130 In a more recent study, the entrapment efficiency of siRNA in PLGA nanoparticles was enhanced by incorporating a cationic polymer, polyethylenimine (PEI), into the PLGA matrix.131 The effectiveness of the siRNA-loaded PLGA-PEI nanoparticles in silencing a model gene, fire-fly luciferase, was investigated in cell culture using the MDA-kb2 cell line. The presence of PEI in the PLGA nanoparticle matrix increased siRNA encapsulation by about 2-fold and was accompanied by an improved siRNA release profile. PLGA-PEI nanoparticles transporting luciferase-targeted siRNA enabled effective silencing of the gene in cells stably expressing luciferase, as well as in cells that could be induced to overexpress the gene. Inclusion of PEI in the PLGA matrix resulted in greater cellular accumulation of the nanoparticles. The positive charge on the nanoparticle surface possibly enabled the nanoparticles to escape the lysosome; both higher uptake and greater cytosolic delivery could have contributed to the gene silencing effectiveness of PLGA-PEI nanoparticles. Serum stability and lack of cytotoxicity add to the potential of these engineered nanoparticles in gene silencing-based therapeutic applications.


COS-7 and Cf2th cells

Human prostate tumors cells

A549 cells (lung cancer) Hepatic cells

Antisense oligonucleotides DNA/RNA DNA

Beta-Gal plasmid

DNA

Luciferase gene

Chitosan-coated PLGA nanoparticles


PLGA nanoparticles by double-emulsion method

Complex PLGA nanoparticles with PEI and ultrasonically directed (high positive zeta potential)

Bioadhesive PLGA nanoparticles stabilized with Carbopol

Cationic lipid 1,2-dioleoyl3-(trimethyl ammonium) propane-modified PLGA nanoparticles conjugated with asialofetuin

Lung cancer cells

Hepatocarcinoma

Thymidine kinase gene plasmid P(EGFP-AFP)

PLGA nanoparticles by double-emulsion method

Target Site/Tested Cells

Specific Gene Delivered


Table 6. PLGA Nanoparticles for Gene Delivery

Increased level of gene in liver cells after intravenous administration; 5- to 12fold increase in transfection efficiency

Higher transfection efficiency compared with the Pluronic-stabilized or necked nanoparticles

Enhanced in vivo tumor cell transfection; 8-fold increase in tumor cell transfection efficacy in irradiated tumors

Localization into the endolysosomal compartment; cell viability was not affected by nanoparticles; 250-fold protein expression in cells

Efficient delivery of antisense oligonucleotides

High entrapment (91.25%) efficiency; enhanced transfection efficiency; efficient protection of plasmid DNA by nuclease


109

167

166

165

126

124

Reference


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VI. ENHANCED ORAL BIOAVAILABILITY OF ANTICANCER DRUGS VIA PLGA NANOPARTICLES FOR CANCER THERAPEUTICS Oral chemotherapy is the most appealing approach for cancer treatment because of its noninvasive nature and therefore better patient compliance. However, most anticancer drugs cannot be delivered orally because of their poor oral bioavailability.132 When these drugs are administered via the oral route, only a fraction of the administered dose is available to the systemic circulation. For example, oral bioavailability of paclitaxel133 and docetaxel are 1% and