Enhancement of cellular uptake and cytotoxicity of curcumin-loaded PLGA nanoparticles by conjugation with anti-P-glycoprotein in drug resistance cancer cells

Aim:

To compare the anti-cancer activity and cellular uptake of curcumin (Cur) delivered by targeted and non-targeted drug delivery systems in multidrug-resistant cervical cancer cells.

Methods:

Cur was entrapped into poly (DL-lactide-co-glycolide) (PLGA) nanoparticles (Cur-NPs) in the presence of modified-pluronic F127 stabilizer using nano-precipitation technique. On the surface of Cur-NPs, the carboxy-terminal of modified pluronic F127 was conjugated to the amino-terminal of anti-P-glycoprotein (P-gp) (Cur-NPs-APgp). The physical properties of the Cur-NPs, including particle size, zeta potential, particle morphology and Cur release kinetics, were investigated. Cellular uptake and specificity of the Cur-NPs and Cur-NPs-APgp were detected in cervical cancer cell lines KB-V1 (higher expression of P-gp) and KB-3-1 (lower expression of P-gp) using fluorescence microscope and flow cytometry, respectively. Cytotoxicity of the Cur-NPs and Cur-NPs-APgp was determined using MTT assay.

Results:

The particle size of Cur-NPs and Cur-NPs-APgp was 127 and 132 nm, respectively. The entrapment efficiency and actual loading of Cur-NPs-APgp (60% and 5 μg Cur/mg NP) were lower than those of Cur-NPs (99% and 7 μg Cur/mg NP). The specific binding of Cur-NPs-APgp to KB-V1 cells was significantly higher than that to KB-3-1 cells. Cellular uptake of Cur-NPs-APgp into KB-V1 cells was higher, as compared to KB-3-1 cells. However, the cellular uptake of Cur-NPs and Cur-NPs-IgG did not differ between the two types of cells. Besides, the cytotoxicity of Cur-NPs-APgp in KB-V1 cells was higher than those of Cur and Cur-NPs.

Conclusion:

The results demonstrate that Cur-NPs-APgp targeted to P-gp on the cell surface membrane of KB-V1 cells, thus enhancing the cellular uptake and cytotoxicity of Cur.     

Comparing cellular uptake and cytotoxicity of targeted drug carriers in cancer cell lines with different drug resistance mechanisms

The purpose of this study was to compare the cellular uptake and cytotoxicity of targeted and nontargeted doxorubicin (DOX)-loaded poly(d,l-lactide co-glycolide) (PLGA) nanoparticle (NP) drug delivery systems in drug-resistant ovarian (SKOV-3) and uterine (MES-SA/Dx5) cancer cell lines. The cellular uptakes of DOX from nonconjugated DOX-loaded NPs (DNPs) and from HER-2 antibody-conjugated DOX-loaded NPs (ADNPs) in MES-SA/Dx5 cancer cells were higher compared to free DOX. Results also showed higher uptake of DOX from ADNPs in SKOV-3 cells compared with both free DOX and DNPs treatment. Cytotoxicity results at 10 μM extracellular DOX concentration were consistent with the cellular uptake results. Our study concludes that cellular uptake and cytotoxicity of DOX can be improved in MES-SA/Dx5 cells by loading DOX into PLGA NPs. DNPs targeted to membrane receptors may enhance cellular uptake and cytotoxicity in SKOV-3 cells. FROM THE CLINICAL EDITOR: The authors of this study compare the cellular uptake and cytotoxicity of targeted and nontargeted doxorubicin loaded PLGA nanoparticle delivery systems in drug-resistant ovarian and uterine cancer cell lines, concluding that cellular uptake and cytotoxicity of doxorubicin can be improved by the proposed methods.     

Biodegradable nanoparticles mimicking platelet binding as a targeted and controlled drug delivery system

This research aims to develop targeted nanoparticles as drug carriers to the injured arterial wall under fluid shear stress by mimicking the natural binding ability of platelets via interactions of glycoprotein Ib-alpha (GPIbα) of platelets with P-selectin of damaged endothelial cells (ECs) and/or with von Willebrand factor (vWF) of the subendothelium. Drug-loaded poly(d,l-lactic-co-glycolic acid) (PLGA) nanoparticles were formulated using a standard emulsion method and conjugated with glycocalicin, the external fraction of platelet GPIbα, via carbodiimide chemistry. Surface-coated and cellular uptake studies in ECs showed that conjugation of PLGA nanoparticles, with GPIb, significantly increased nanoparticle adhesion to P-selectin- and vWF-coated surfaces as well as nanoparticle uptake by activated ECs under fluid shear stresses. In addition, effects of nanoparticle size and shear stress on adhesion efficiency were characterized through parallel flow chamber studies. The observed decrease in bound nanoparticle density with increased particle sizes and shear stresses is also explained through a computational model. Our results demonstrate that the GPIb-conjugated PLGA nanoparticles can be used as a targeted and controlled drug delivery system under flow conditions at the site of vascular injury.     

Brain-targeted delivery of paclitaxel using glutathione-coated nanoparticles for brain cancers

Paclitaxel is not effective for treatment of brain cancers because it cannot cross the blood–brain barrier (BBB) due to efflux by P-glycoprotein (P-gp). In this work, glutathione-coated poly-(lactide-co-glycolide) (PLGA) nanoparticles (NPs) of paclitaxel were developed for brain targeting for treatment of brain cancers. P-gp ATPase assay was used to evaluate the NP as potential substrates. The NP showed a particle size suitable for BBB permeation (particle size around 200?nm) and higher cellular uptake of the NP was demonstrated in RG2 cells. The P-gp ATPase assay suggested that the NP were not substrate for P-gp and would not be effluxed by P-gp present in the BBB. The in vitro release profile of the NP exhibited no initial burst release and showed sustained drug release. The proposed coated NP showed significantly higher cytotoxicity in RG2 cells compared with uncoated NP (p?≤?0.05). Tubulin immunofluorescent study showed higher cell death by the NP due to increased microtubule stabilization. In vivo brain uptake study in mice showed higher brain uptake of the NP containing coumarin-6 compared with solution. The proposed brain-targeted NP delivery of paclitaxel could be an effective treatment for the brain cancers.     

Biochemical mechanism of modulation of human P-glycoprotein (ABCB1) by curcumin I, II, and III purified from Turmeric powder

P-glycoprotein (Pgp, ABCB1) is an ATP-dependent drug efflux pump linked to development of multidrug resistance (MDR) in cancer cells. Previously [Biochem Pharmacol 2002;64:573-82], we reported that a curcumin mixture could modulate both function and expression of Pgp. This study focuses on the effect of three major curcuminoids--curcumin I, II and III purified from a curcumin mixture--on modulation of Pgp function in a multidrug resistant human cervical carcinoma cell line (KB-V1). The similar IC(50) values for cytotoxicity of curcuminoids of KB-V1, and KB-3-1 (parental drug sensitive cell line) suggest that these curcuminoids may not be substrates for Pgp. Treating the cells with non-toxic doses of curcuminoids increased their sensitivity to vinblastine only in the Pgp expressing drug resistant cell line, KB-V1, and curcumin I retained the drug in KB-V1 cells more effectively than curcumin II and III, respectively. Effects of each curcuminoid on rhodamine123, calcein-AM, and bodipy-FL-vinblastine accumulation confirmed these findings. Curcumin I, II and III increased the accumulation of fluorescent substrates in a dose-dependent manner, and at 15 microM, curcumin I was the most effective. The inhibitory effect in a concentration-dependent manner of curcuminoids on verapamil-stimulated ATPase activity and photoaffinity labeling of Pgp with the [(125)I]-iodoarylazidoprazosin offered additional support; curcumin I was the most potent modulator. Taken together, these results indicate that curcumin I is the most effective MDR modulator among curcuminoids, and may be used in combination with conventional chemotherapeutic drugs to reverse MDR in cancer cells.     

Surfactant-polymer nanoparticles overcome P-glycoprotein-mediated drug efflux

Nanoparticles enhance the therapeutic efficacy of an encapsulated drug by increasing and sustaining the delivery of the drug inside the cell. We have previously demonstrated that Aerosol OT (AOT)-alginate nanoparticles, a novel formulation developed recently in our laboratory, significantly enhance the therapeutic efficacy of encapsulated drugs like doxorubicin in drug-sensitive tumor cells. The purpose of this study is to evaluate the drug delivery potential of AOT-alginate nanoparticles in drug-resistant cells overexpressing the drug efflux transporter, P-glycoprotein (P-gp). AOT-alginate nanoparticles were formulated using an emulsion-cross-linking process. Rhodamine 123 and doxorubicin were used as model P-gp substrates. Cytotoxicity of nanoparticle-encapsulated doxorubicin and kinetics of nanoparticle-mediated cellular drug delivery were evaluated in both drug-sensitive and -resistant cell lines. AOT-alginate nanoparticles enhanced the cytotoxicity of doxorubicin significantly in drug-resistant cells. The enhancement in cytotoxicity with nanoparticles was sustained over a period of 10 days. Uptake studies with rhodamine-loaded nanoparticles indicated that nanoparticles significantly increased the level of drug accumulation in resistant cells at nanoparticle doses higher than 200 microg/mL. Blank nanoparticles also improved rhodamine accumulation in drug-resistant cells in a dose-dependent manner. Nanoparticle-mediated enhancement in rhodamine accumulation was not because of membrane permeabilization. Fluorescence microscopy studies demonstrated that nanoparticle-encapsulated doxorubicin was predominantly localized in the perinuclear vesicles and to a lesser extent in the nucleus, whereas free doxorubicin accumulated mainly in peripheral endocytic vesicles. Inhibition of P-gp-mediated rhodamine efflux with AOT-alginate nanoparticles was confirmed in primary brain microvessel endothelial cells. In conclusion, an AOT-alginate nanoparticle system enhanced the cellular delivery and therapeutic efficacy of P-gp substrates in P-gp-overexpressing cells.     

Brain targeted delivery of paclitaxel using endogenous ligand

The objective of the present investigation was to formulate nanoparticles constructed using PLGA polymer for the effective targeted delivery to brain via nasal route. The PLGA nanoparticles were optimized using novel design of experiment technique by 2³ full factorial design. Drug: polymer ratio (X₁), surfactant concentration (X₂) and stirring speed (X₃) were identified as critical process parameters, and its impact on particle size (Y₁) and % entrapment efficiency (Y₂) was studied. The optimized nanoparticle formulation was conjugated with glutathione as an endogenous ligand by using carbodiimide chemistry using (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) (EDAC) as linker molecule. From Ellman's assay, it was found that a total of 691.27 ± 151 units of glutathione were conjugated upon each PLGA nanoparticle. The in vitro release studies as well as ex vivo studies revealed biphasic pattern of drug release with initial burst release followed by slow exponential release of drug over a period of 24 h. The in vivo biodistribution studies were conducted on rat following nasal administration of the nanoparticle formulation (conjugated and unconjugated) and were compared with plain paclitaxel suspension. The results clearly demonstrated that the brain targeting efficiency was enhanced with the glutathione conjugated formulation (387.474%) as compared to the unconjugated nanoparticle formulation (224.327%). Further, the in vitro in vivo correlation studies revealed good relationship (R² > 0.95) as obtained from the levy plot. Glutathione proves to be an efficient vector for the successful transport of poor bioavailable drug to the brain.     

Evaluation of nanoparticles loaded with benzopsoralen in rat peritoneal exudate cells

Psoralens are widely used for the treatment of hyperproliferative skin disease. In this work, we prepared nanoparticles (NP) containing a benzopsoralen (3-ethoxy carbonyl-2H-benzofuro[3,2-f]-1-benzopiran-2-one) by the solvent evaporation technique. We evaluated important NP parameters such as particle size, drug encapsulation efficiency, effect of the encapsulation process over the drug's photochemistry, zeta potential, external morphology, and in vitro release behavior. We also investigated the nanoparticle as a drug delivery system (DDS), as well as its target delivery to the action site, which is a very important parameter to increase the therapeutic use of psoralens and to reduce their side effects. The uptake of benzopsoralen-loaded PLGA nanoparticles by different kinds of cells found in rat peritoneal exudates was also studied. The photodamage promoted by irradiation with UV light revealed morphological characteristics of cell damage such as cytoplasmic vesiculation, mitochondrial damage, and swelling of both the granular endoplasmatic reticulum and nuclear membrane. This encapsulation method maintained the drug's properties and improved drug delivery to the target cell.     

Biochemical mechanism of modulation of human P-glycoprotein by stemofoline

The resistance to chemotherapeutic drugs by cancer cells is considered to be one of the major obstacles for success in the treatment of cancer. A major mechanism underlying this multidrug resistance is the overexpression of P-glycoprotein (P-gp), resulting in insufficient drug delivery to the tumor sites. A previous study has shown that stemofoline, an alkaloid isolated from Stemona burkillii, could enhance the sensitivity of chemotherapeutics in a synergistic fashion. In the present study, we have focused on the effect of stemofoline on the modulation of P-gp function in a multidrug resistant human cervical carcinoma cell line (KB-V1). The effects of stemofoline on a radiolabeled drug, [(3)H]-vinblastine, and fluorescent P-gp substrates, rhodamine 123 and calcein-AM accumulation or retention were investigated to confirm this finding. Stemofoline could increase the accumulation or retention of radiolabeled drugs or fluorescent P-gp substrates in a dose-dependent manner. For additional studies on drug-P-gp binding, P-gp ATPase activity was stimulated by stemofoline in a concentration-dependent manner. More evidence was offered that stemofoline inhibits the effect on photoaffinity labeling of P-gp with [(125)I]-iodoarylazidoprazosin in a concentration-dependent manner. These data indicate that stemofoline may interact directly with P-gp and inhibit P-gp activity, whereas stemofoline has no effect on P-gp expression. Taken together, the results exhibit that stemofoline possesses an effective MDR modulator, and may be used in combination with conventional chemotherapeutic drugs to reverse MDR in cancer cells.     

Size-dependency of nanoparticle-mediated gene transfection: studies with fractionated nanoparticles

Nanoparticles formulated from biodegradable polymers such as poly (lactic acid) and poly (D,L-lactide-co-glycolide) (PLGA) are being extensively investigated as non-viral gene delivery systems due to their sustained release characteristics and biocompatibility. PLGA nanoparticles for DNA delivery are mainly formulated using an emulsion-solvent evaporation technique. However, this formulation procedure results in the formation of particles with heterogeneous size distribution. The objective of the present study was to determine the relative transfectivity of the smaller- and the larger-sized fractions of nanoparticles in cell culture. PLGA nanoparticles containing a plasmid DNA encoding luciferase protein as a marker were formulated by a multiple emulsion-solvent evaporation method (mean particle diameter = 97 +/- 3 nm) and were fractionated using a membrane (pore size: 100 nm) filtration technique. The particles that passed through the membrane were designated as the smaller-sized nanoparticles (mean diameter = 70 +/- 2 nm) and the fraction that was retained on the membrane as the larger-sized nanoparticles (mean diameter = 202 +/- 9 nm). The smaller-sized nanoparticles showed a 27-fold higher transfection than the larger-sized nanoparticles in COS-7 cell line and a 4-fold higher transfection in HEK-293 cell line. The surface charge (zeta potential), cellular uptake, and the DNA release were almost similar for the two fractions of nanoparticles, suggesting that some other yet unknown factor(s) is responsible for the observed differences in the transfection levels. The results suggest that the particle size is an important factor, and that the smaller-sized fraction of the nanoparticle formulation predominantly contributes towards their transfection.     

Design of a Multifunctional PLGA Nanoparticulate Drug Delivery System: Evaluation of its Physicochemical Properties and Anticancer Activity to Malignant Cancer Cells

Purpose  Several individual approaches were combined to fabricate a novel nanoparticulate drug delivery system to achieve targeting and anticancer effects in various malignant cancer cells. Methods  Doxorubicin was conjugated to Poly(lactic-co-glycolic acid) (PLGA), which was formulated into nanoparticle via solvent-diffusion method. The surface of the nanoparticles was subsequently linked with Poly(ethylene glycol) (PEG) and Arg-Gly-Asp (RGD) peptide to realize both passive and active targeting functions. The multifunctional nanoparticles were then tested against several malignant cancer cell lines. Results  The conjugation increased loading efficiency of doxorubicin to PLGA nanoparticles (the encapsulation efficiency was over 85%) and alleviated the drug burst release effect substantially. The drug was released from the polymeric matrix in a sustained release manner over a period of 12 days. The resultant nanoparticles were spherically uniform and well-dispersed. The nanoparticle targeting ability was proven through strong affinity to various integrin-expressing cancer cells, and much less affinity to the low integrin expression cancer cells. The nanoparticles also showed high efficacy in inducing apoptosis in specific malignant cancer cell. Conclusion  The developed multifunctional nanoparticles hold potential to treat malignant integrin-expressing cancers.     

Novel flavonoid-based biodegradable nanoparticles for effective oral delivery of etoposide by P-glycoprotein modulation: an in vitro,ex vivo and in vivo investigations

Abstract

A receptor level interaction of etoposide with P-glycoprotein (P-gp) and subsequent intestinal efflux has an adverse effect on its oral absorption. The present work is aimed to enhance the bioavailability of etoposide by co-administering it with quercetin (a P-gp inhibitor) in dual-loaded polymeric nanoparticle formulation. Poly-lactic-co-glycolic acid (PLGA) nanoparticles were optimized for various parameters like o/w phase volume ratio, poly-vinyl alcohol concentration, PLGA concentration and sonication time. The cytotoxicity studies (MTT assay) revealed a 9- and 11-fold decrease in the IC 50 values for etoposide-loaded nanoparticles (ENP) and etoposide?+?quercetin dual-loaded nanoparticles (EQNP) when compared to that of free etoposide, respectively, and the results were further supported by florescent-activated cell sorter studies. The confocal imaging of the intestinal sections treated with ENP and EQNP containing fluorescent probe (rhodamine) showed the superiority of the EQNP to permeate deeper. Furthermore, pharmacokinetic studies on rats revealed that EQNP exhibited a 2.4-fold increase in bioavailability of etoposide than ENP with no quercetin. The developed loaded nanoparticles have the high potential to enhance the bioavailability of the etoposide and sensitize the resistant cells.     

Susceptibility of nanoparticle-encapsulated paclitaxel to P-glycoprotein-mediated drug efflux

Overexpression of P-glycoprotein (P-gp) is a key factor contributing to the development of multidrug resistance (MDR) in cancer cells. The objective of the study is to investigate whether a P-gp substrate, paclitaxel, delivered to MDR tumor cells in poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles is susceptible to P-gp – mediated drug efflux. Paclitaxel-loaded nanoparticles were formulated by emulsion-solvent evaporation technique. Nanoparticles had a mean hydrodynamic diameter of about 195 nm, and demonstrated sustained release of paclitaxel. In vitro cell culture studies indicated that paclitaxel nanoparticles result in sustained, dose-dependent and significant cytotoxicity in drug-sensitive MCF-7 tumor cells but not in drug-resistant NCI-ADR/RES cells. Resistance to nanoparticle-encapsulated paclitaxel was reversed by verapamil, a P-gp inhibitor. Further, sustained inhibition of P-gp was necessary for sustaining the cytotoxicity of nanoparticle-encapsulated paclitaxel in drug-resistant cells. Inhibition of P-gp by verapamil did not significantly affect the uptake or retention of nanoparticles in drug-resistant cells. In conclusion, our studies suggest that P-gp substrates, such as paclitaxel, delivered to MDR cells by PLGA nanoparticles, are susceptible to efflux by P-gp. Inhibition of P-gp restores sensitivity to paclitaxel; however, sustained inhibition of P-gp is required for sustained therapeutic efficacy of nanoparticle-encapsulated drug.     

Synergism of energy starvation and dextran-conjugated doxorubicin in the killing of multidrug-resistant KB carcinoma cells

Here we report that 2-deoxyglucose/Na azide treatment and free/conjugated doxorubicin are synergistic in cell killing. As demonstrated by fluorescence confocal microscopy, KB-V1 cells retained more conjugated doxorubicin than free doxorubicin. Verapamil or 2-deoxyglucose/Na azide enhanced only the retention of the free drug and the small (<70 kDa) conjugates, indicating that P-glycoprotein (P-gp) is not effective against large conjugates. Conjugated doxorubicin was excluded from nuclei. Initially both free and conjugated doxorubicin accumulated in cytoplasmic organelles. Upon 2-deoxyglucose/Na azide treatment, fluorescence labeling of organelles dissipated. Prolonged (24 h) incubation of conjugate-preloaded cells resulted in redistribution of some of the organelle-associated fluorescence to nuclei, suggesting decoupling. The appearance of free doxorubicin was confirmed by capillary electrophoresis. 2-Deoxyglucose/Na azide treatment also retarded decoupling. Our results suggest that energy starvation, in addition to increasing cellular retention of P-gp substrates, may affect cellular fate of conjugated drugs with a possible enhancing effect in cancer cell killing.     

Nanoglue: an alternative way to display cell-internalizing Peptide at the spikes of hepatitis B virus core nanoparticles for cell-targeting delivery

Cell-internalizing peptides (CIPs) can be used to mediate specific delivery of nanoparticles across cellular membrane. The objective of this study was to develop a display technique using hepatitis B virus (HBV) capsid-binding peptide as a "nanoglue" to present CIPs on HBV nanoparticles for cell-targeting delivery. A CIP was selected from a phage display library and cross-linked specifically at the tips of the spikes of the HBV capsid nanoparticle via the "nanoglue" by using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS). Fluorescent oligonucleotides packaged in the nanoparticles and the fluorescein molecules conjugated on the nanoparticles were delivered to cells by using this display technique. This study demonstrated a proof of principle for cell-targeting delivery via "nanoglue" bioconjugation.     

Effects of aggregation and the surface properties of gold nanoparticles on cytotoxicity and cell growth

The effect of gold nanoparticles (Au NPs) on cells remains open for investigation. Here we show that small Au NPs can be endocytosed by cells and form aggregates inside the cell, resulting in cytotoxicity. When the aggregates become too large to enter the cell and instead adhere onto the cell surface, however, the growth rate of HeLa cells increases. Printed patterns of Au NPs fabricated through inkjet printing technology were used to study the effects of Au NP aggregation on human cervical carcinoma (HeLa) cell activity. The growth of the HeLa cells was inhibited on the polymer-coated Au NPs but increased on the silicon substrate. On the uncoated Au NP surface, however, the HeLa cell growth rate was higher than that on the silicon substrate. Experiments with Escherichia coli cells showed a similar effect of the Au NPs. This phenomenon provides a new perspective for research on toxicity in nanoparticle biology. FROM THE CLINICAL EDITOR: Printed patterns of Au NPs fabricated through inkjet printing technology were used to study the effects of Au NP aggregation on human cervical carcinoma (HeLa) cell activity. Small Au NPs can be endocytosed by cells resulting in cytotoxicity; in contrast, large aggregates adhere onto the cell surface and increase the growth rate of HeLa cells.     

SS-31 peptide enables mitochondrial targeting drug delivery: a promising therapeutic alteration to prevent hair cell damage from aminoglycosides

Aminoglycoside-induced hearing loss stems from damage or loss of mechanosensory hair cells in the inner ear. Intrinsic mitochondrial cell death pathway plays a key role in that cellular dysfunction for which no proven effective therapies against oto-toxicities exist. Therefore, the aim of the present study was to develop a new mitochondrial targeting drug delivery system (DDS) that provided improved protection from gentamicin. Particularly, SS-31 peptide-conjugated geranylgeranylacetone (GGA) loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles were constructed successfully via emulsion-solvent evaporation method. The zebrafish lateral line sensory system was used as an in vivo evaluating platform to investigate the protective efficiency against gentamicin. SS-31 modification significantly reduced the activity of mechanoelectrical transduction (MET) channel and gentamicin uptake in zebrafish lateral line hair cells. As expected, SS-31 conjugated nanoparticles showed mitochondrial specific accumulation in hair cells when compared with unconjugated formulations. Furthermore, intracellular SS-31 modified PLGA NPs slightly enhanced mitochondrial membrane potential (MMP, ΔΨ_m) and then returned to a steady-state, indicating their effect on the respiratory chain complexes in mitochondria. GGA loaded SS-31 conjugated nanoparticles demonstrated the most favorable hair cells survivals against gentamicin when compared with unconjugated groups whereas blank formulations failed to exhibit potency, indicating that the efficiency was attributed to drug delivery of GGA. These results suggest that our constructed mitochondria-targeting PLGA based DDS have potential application in protecting hair cells from ototoxic agents.     

Polymeric nanoparticles for siRNA delivery and gene silencing

Gene silencing using small interfering RNA (siRNA) has several potential therapeutic applications. In the present study, we investigated nanoparticles formulated using the biodegradable polymer, poly(d,l-lactide-co-glycolide) (PLGA) for siRNA delivery. A cationic polymer, polyethylenimine (PEI), was incorporated in the PLGA matrix to improve siRNA encapsulation in PLGA nanoparticles. PLGA-PEI nanoparticles were formulated using double emulsion-solvent evaporation technique and characterized for siRNA encapsulation and in vitro release. The effectiveness of siRNA-loaded PLGA-PEI nanoparticles in silencing a model gene, fire-fly luciferase, was investigated in cell culture. Presence of PEI in PLGA nanoparticle matrix increased siRNA encapsulation by about 2-fold and also improved the siRNA release profile. PLGA-PEI nanoparticles carrying 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. Quantitative studies indicated that presence of PEI in PLGA nanoparticles resulted in 2-fold higher cellular uptake of nanoparticles while fluorescence microscopy studies showed that PLGA-PEI nanoparticles delivered the encapsulated siRNA in the cellular cytoplasm; 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 further add to the potential of PLGA-PEI nanoparticles in gene silencing-based therapeutic applications.     

Anticancer activity of cisplatin-loaded PLGA-mPEG nanoparticles on LNCaP prostate cancer cells.

The in vitro anticancer activity of cisplatin-loaded PLGA-mPEG nanoparticles on human prostate cancer LNCaP cells was investigated. The uptake of the PLGA-mPEG nanoparticles by the LNCaP cells was also studied. Blank PLGA-mPEG nanoparticles exhibited low cytotoxicity, which increased with increasing PLGA/PEG ratio in the PLGA-mPEG copolymer used to prepare the nanoparticles, possibly due to the increased cell uptake observed with increasing PLGA/PEG ratio. PLGA-mPEG nanoparticles loaded with cisplatin exerted in vitro anticancer activity against LNCaP cells that was comparable to the activity of free (non-entrapped in nanoparticles) cisplatin. Little differences in the in vitro anticancer activity of the different nanoparticle compositions were found, which may result from the differences observed between the different nanoparticles compositions in the uptake by the LNCaP cells and in the leakage of cisplatin from the nanoparticles during incubation with the cells. Visual evidence of nanoparticles' uptake by the LNCaP cells was obtained with nanoparticles labeled with PLGA(4165)-PyrBu(274) or dextran-rhodamine B isothiocyanate using fluorescence microscopy. Moreover, in some cases fluorescence around or inside cell nuclei was observed, which, if verified by further studies, would indicate that PLGA-PEG nanoparticles might prove to be useful in site-specific delivery of drugs whose site of pharmacological activity is cell nucleus.