Intracellular Delivery of Saquinavir in Biodegradable Polymeric Nanoparticles for HIV/AIDS
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- Shah, L.K. & Amiji, M.M. Pharm Res (2006) 23: 2638. doi:10.1007/s11095-006-9101-7
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This study aims at developing poly(ethylene oxide)-modified poly(epsilon-caprolactone) (PEO-PCL) nanoparticulate system as an intracellular delivery vehicle for saquinavir, an anti-HIV protease inhibitor.
Materials and Methods
Saquinavir-loaded PEO-PCL nanoparticles were prepared by a solvent displacement process. The formed nanoparticles were characterized for size, surface charge, and surface presence of PEO chains. Cellular uptake and distribution of the nanoparticle was examined in THP-1 human monocyte/macrophage (Mo/Mac) cell line. Intracellular saquinavir concentrations were measured as a function of dose and duration of incubation.
The PEO-PCL nanoparticles had a smooth surface and spherical shape and showed a relatively uniform size distribution with a mean particle diameter of approximately 200 nm. The surface presence of PEO chains was confirmed by an increase in the –C–O–(ether) signature of the C1s spectra in electron spectroscopy for chemical analysis. Rapid cellular uptake of rhodamine-123 encapsulated PEO-PCL nanoparticles was observed in THP-1 cells. Intracellular saquinavir concentrations when administered in the nanoparticle formulation were significantly higher than from aqueous solution.
This study shows that PEO-PCL nanoparticles provide a versatile platform for encapsulation of saquinavir and subsequent intracellular delivery in Mo/Mac cells.
Key wordsintracellular delivery nanoparticles poly(ethylene oxide)-modified poly(epsilon-caprolactone) saquivanir THP-1 monocytes/macrophages
Therapeutic use of anti-HIV protease inhibitors suffers from problems of poor solubility, low and variable oral bioavailability with limited penetration into lymphatic and central nervous systems (1, 2, 3). The high cost of therapy has added to the cause of significant crisis in the management of HIV/AIDS patients, especially in developing nations. A major barrier to the current therapy is the development of resistance due to the persistence of HIV in the sanctuary sites where the virus thrives (4). A paradigm shift in the last couple of decades has led to utilization of targeted delivery mechanism for various therapeutic agents in order to increase efficacy and reduce toxicity (5). Anti-HIV therapy may explore novel drug delivery strategies as a new dimension along with multiple drug therapy (the highly active antiretroviral therapy, HAART).
One of the recent trends has been the use of nanocarrier delivery technology, where the payload is trapped within a carrier system of 1 to 1,000 nm in diameter (6,7). Nanocarriers, due to their small size and target specific localization properties, are being actively investigated for preferential drug delivery to various disease sites in the body. Higher concentrations and increased residence time of the drugs can be achieved at the sanctuary sites and thus help to reduce the viral load significantly (8). Regardless of the inherent properties of the drug candidates, the pharmacokinetics and distribution pattern will be dictated by the properties of the nanocarrier system (9). Different types of nanoparticle systems can be fabricated for enhancing oral absorption as well as transport across the blood–brain barrier by either passive diffusion or by carrier-mediated endocytosis (10). For instance, Boudad et al. (11) have prepared hydroxypropyl-beta-cyclodextrin inclusion complex of saquinavir and encapsulated in poly(alkylcyanoacrylate) nanoparticles. With an aim to increase the intracellular concentration of saquinavir, we have developed a stable formulation using poly(ethylene oxide)-modified poly(epsilon-caprolactone) (PEO-PCL) nanoparticulate system and characterized it. PCL is a synthetic pre-formed biodegradable polymer. It possesses unique properties such as excellent biocompatibility, high hydrophobicity, and neutral biodegradation end products, which do not disturb the pH balance of the degradation medium. It is a crystalline polymer and degrades very slowly in vitro in the absence of enzymes and in vivo as well (12). PCL has a melting range of 59–64°C, depending on its crystalline state. The crystallinity of PCL varies with its molecular weight and plays an important role in determining both permeability and biodegradability because of the fact that bulk crystalline phase is inaccessible to water and other permeating agents (13). An increase in crystallinity reduces the permeability by reducing the solubility of the drug and increasing the tortuosity of the diffusional pathway. The biodegradation rate is also significantly reduced by the decrease in accessibility of the ester bond (14). High permeability to many drugs and a lack of toxicity has made PCL and its derivatives well suited for colloidal drug delivery (14,15).
Surface modification of PCL nanoparticles with PEO chains prevents aggregation and allows for efficient systemic delivery especially after intravenous administration. Pluronics® (also known as poloxamers) are non-ionic ABA-type triblock copolymers with the general formula HO(C2H4O)a(C3H6O)b(C2H4O)aH, consisting of two PEO chains and a center block made of poly(propylene oxide) (PPO). They are commercially available in different grades and vary from liquids, semisolids, to solids based on the ratio of PEO to PPO and the molecular weight. Pluronics have been used as emulsifying agents, solubilizing agents, surfactants, and wetting agents. As the “a” and “b” block numbers change, hydrophilic–lipophilic balance changes, thereby changing the physical chemical properties of the triblock copolymer, changing its applicability (15). Pluronic® F-108/NF (poloxamer 338) has a bulky central block as well as long side arms (a=122; b=56).
Saquinavir, the first HIV-protease inhibitor to be marketed as Invirase® for the treatment of HIV, is a peptide derivative that inhibits HIV-1 and HIV-2 protease-mediated cleavage of the gag and pol polyproteins of the HIV genome, thus preventing the post-translational processing required for virus maturation and spread (16). The oral bioavailability of a single dose of saquinavir is only 4% (16, 17, 18). Its low bioavailability is mainly attributed to its metabolism by cytochrome P450 3A4 and to the membrane transporters namely the P-glycoprotein (P-gp) (4,19). After performing the loading and release studies, intracellular uptake studies were carried out in monocyte/macrophage (Mo/Mac) cells. It has been known that the HIV infects peripheral mononuclear phagocytic cells and the central nervous system in the early stages (19). The phagocytic cells (Mo/Mac) also act as a shuttle for the viruses to go to other sites in the body and thrive there (2). Some of the phagocytic cells also express P-gp efflux pump, which is known to present a therapeutic limitation by preventing the penetration and retention of anti-HIV protease inhibitors.
Bender et al. (20) have showed that at a concentration of 100 nM, free saquinavir was completely inactive in chronically HIV-infected Mo/Mac cells, but when bound to poly(hexylcyanoacrylate) nanoparticles, caused a 35% decrease in virus production. We hypothesize that the Mo/Mac would endocytose the nanoparticulate formulation and provide optimal concentrations in Mo/Mac cells as well as transport the drug to CNS and other sites, which are a potential reservoirs for the HIV. The drug would be released at these sites and help in targeted and effective eradication of the viral load. The preliminary studies shown here revealed that PEO-PCL nanoparticulate system is an efficient delivery vehicle for the release of anti-HIV protease inhibitor and its intracellular delivery to the Mo/Mac cells.
Materials and Methods
Saquinavir base, a protease inhibitor with molecular weight of 670.9 Da, was purchased from Aapin Chemicals, (London, UK). PCL of molecular weight of 14,800 Da., as confirmed by gel electrophoresis, was purchased from Polysciences, (Warrington, PA). National Formulary grade Pluronic® F-108 was kindly supplied by the Performance Chemical Division of BASF Inc., (Parsippany, NJ). Rhodamine-123 was obtained from Molecular Probes (Eugene, OR). Lipase (90 U/g) was purchased from ICN Biochemicals, (Aurora, OH). THP-1 human monocyte/macrophage cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA). Deionized distilled water (NanoPureII, Barnstead, and Dubuque, IA) was used for the preparation of aqueous solutions. All other reagents were purchased from Fischer Scientific.
Preparation of PEO-PCL Nanoparticles
The PEO-PCL nanoparticles were prepared by a solvent displacement method similar to the one used by Chawla et al. (14). Briefly, 850 mg of PCL and 150 mg of Pluronic® F-108NF were dissolved in 30-ml acetone by mild heating. The polymer solution was added drop wise into 200 ml of deionized distilled water under continuous stirring over a magnetic stirrer. Stirring was continued overnight to evaporate the organic solvent. The resulting suspension of nanoparticles was centrifuged at 10,000×g for 20 min. The supernatant was discarded and pellet washed twice with deionized distilled water and freeze dried. Saquinavir (5% w/w)-loaded nanoparticles were prepared by a similar procedure as described above, except a known quantity of saquinavir was dissolved in acetone and added to the PCL and Pluronic® F108 solution before addition into aqueous medium thereby encapsulating the drug with the solid polymeric matrix formed.
Characterization of the Nanoparticles
Particle Size Analysis
A sample of nanoparticles suspension prepared was immediately analyzed for particle size. The suspension was diluted suitably in deionized distilled water, sonicated for 1 min and the particle size was determined for unloaded and saquinavir-loaded nanoparticles with a 90Plus ZetaPALS particle sizer (Brookhaven Instruments, Holtsville, NY).
Measurements of the Surface Charge
A suitably diluted aqueous suspension of nanoparticles was mounted in a ZePALS zeta potential analyzer (Brookhaven Instruments, Holtsville, NY) and the mean zeta potential was measured for unloaded and saquinavir-loaded nanoparticles, suspended in deionized distilled water.
Scanning Electron Microscopy (SEM)
Surface morphology of freeze dried sample was observed. A sample of freeze-dried nanoparticles was diluted with deionized distilled water and mounted on an aluminum sample mount and sputter-coated with a gold-palladium alloy to minimize surface charging. SEM was performed using Hitachi S-4800 environmental scanning electron microscope (Hitachi Instruments, San Jose, CA) at an accelerating voltage of 3 kV.
Electron Spectroscopy for Chemical Analysis (ESCA) Studies
Surface modification of the nanoparticles with Pluronic® F108 allows for physical association of the PCL with poly(propylene oxide) block, while leaving the two flanking PEO chains extending out into the surrounding medium. The surface presence of PEO chains was confirmed with ESCA. ESCA was performed on the control and PEO-modified nanoparticle samples at the National ESCA and Surface Analysis for Biomedical Applications Center (NESAC/BIO), University of Washington in Seattle, WA. The spectra of freeze-dried samples were recorded on the spectrophotometer. Standard Scofield photoemission cross-sections were used to determine the surface elemental composition. Identification of chemical functional groups was obtained from the high-resolution peak analysis of the carbon 1s (C1s) envelopes.
Saquinavir Incorporation and In Vitro Release Studies
Saquinavir Loading Studies
High performance liquid chromatography (HPLC) analysis of saquinavir was carried out with Waters 2695 Separations Module using the slightly modified method as described by Janoly et al. (22). Stationary phase was a Zorbax SB-C18 column (4.6×150 mm, 5 μm) and the mobile phase consisted of acetonitrile, tetrahydrofuran, and 0.1-M dihydrogen phosphate buffer (pH 4.0) in a 32:10:58 volume ratio. The sample injection volume was 60 μl and flow-rate of mobile phase was 1.0 ml/min. Detection of saquinavir was performed at 238 nm using a UV detector. For the loading studies, a pre-determined volume of saquinavir solution was added to PCL-Pluronic® F108 solution in acetone. Nanoparticles were prepared as described above and freeze dried. A known amount of nanoparticles was re-dissolved in acetone and subsequent dilutions were made with methanol. The amount of drug loaded per milligram of nanoparticles (i.e., loading capacity) and percent drug loading (i.e., loading efficiency) were determined from a calibration curve of the drug in methanol.
In Vitro Saquinavir Release Studies
The in vitro release of saquinavir was carried out in phosphate-buffered saline (PBS, pH 7.4). Since the drug is insoluble in this medium, 0.5% (w/v) of Tween® 80 was added to enhance the solubility. Tween® 80 also prevents adsorption of the drug to the container surfaces. The drug release from the formulations was performed by adding 15.0 mg of saquinavir nanoparticles in 15.0 ml of the release medium and kept in a rocker maintained at 37°C. At various time intervals, the tubes were centrifuged at 10,000×g for 10 min. Carefully, 2 ml of supernatant was withdrawn and replaced by 2.0 ml of fresh medium in order to maintain sink conditions. The supernatant was filtered through a 0.45-μm filter and the concentration of the drug was determined by HPLC assay. Release studies were performed in absence and presence of the enzyme Pseudomonas lipase, which is known to enhance the degradation of the polymer (14). Cumulative amount and percent saquinavir released from the nanoparticles was calculated from a calibration curve of the drug in Tween®80-containing PBS.
Uptake and Intracellular Distribution of Nanoparticles in Mo/Mac Cells
THP-1 human monocyte/macrophage, a myelomonocytic cell line derived from the blood of a 1-year-old boy with acute monocytic leukemia (23) were maintained in RPMI® 1640 growth medium and incubated at 37°C under 5% CO2 atmosphere. They were sub-cultured four times during the experimental time period. Cell viability was periodically examined using the Trypan blue exclusion assay for the entire duration of the study.
Cell Uptake and Distribution Studies
Rhodamine123, a hydrophobic fluorescent dye was encapsulated in PEO-PCL nanoparticles at 0.4% (w/w) concentration. The dye-containing nanoparticles were prepared exactly similar to those described above and characterized for particle size and charge. The nanoparticles were re-suspended in serum-free media to a 1 mg per mL concentration. Twenty microliter of this suspension was added to approximately 20,000 THP-1 cells per well in a six-well microplate. Cells were incubated for 1 h and directly viewed with a Nikon TE-2000U scanning fluorescence confocal microscope (Melville, NY) at original magnification of 60X and Adobe Photoshop and Image-J software were used for digitization and processing.
Intracellular Saquinavir Concentration Measurements
Preparation of [3H]-Saquinavir Loaded PEO-PCL Nanoparticles
Tritiated [3H]-saquinavir loaded nanoparticles were prepared by incorporating 4.6 μCi of labeled saquinavir (Moravek Biochemicals (Brea, CA) to 20.0 mg of cold saquinavir and following the procedure as described above. The nanoparticles obtained in aqueous suspension were used for further experiments.
Evaluation of Intracellular Drug Concentrations as a Function of Dose
Tritiated [3H]-saquinavir-loaded PEO-PCL nanoparticles in suspension were incubated with the THP-1 cells suspended in serum-free cell culture media, at different doses of saquinavir ranging from 10 to 250 nM. After drug incubation, the media was changed back to RPMI® 1640. Blank nanoparticles and 3H-saquinavir aqueous solution at the above mentioned concentrations served as controls. After a pre-determined time interval of 6 h, the suspension of cells were subjected to centrifugation and cell pellet obtained was washed with sterile PBS to ensure removal of extracellular drug, re-suspended in PBS, lysed with Triton X-100, and the intracellular drug concentration determined by liquid scintillation counting. All the cell lysate were collected in pre-labeled scintillation vials. To each milliliter of the cell lysate, 10 ml of the ScintiSafe Econo® (scintillation cocktail) was added and the samples were allowed to quench for 4 h in the dark before measuring with a liquid scintillation analyzer. The protein content in the cell pellet was determined using the NanoOrange® Protein Quantitation Kit obtained from Molecular Probes (Eugene, Oregon). Saquinavir concentrations in the cell were calculated as nanomolar (nM) of drug per milligram (mg) of protein.
Evaluation of Intracellular Drug Concentrations as a Function of Time
Following a similar protocol as above, intracellular drug concentrations as a function of time were evaluated by incubating cells with a 50‐nM dose of 3H-saquinavir labeled nanoparticles and 3H-saquinavir solution. After several pre-determined time intervals (0.5 to 12 h), the cells were centrifuged, washed, lysed and assayed for drug as described in Section Evaluation of Intracellular Drug Concentrations as a Function of Dose above.
Results and Discussion
Nanoparticulate Systems for Intracellular Drug Delivery
Previous studies from our lab have shown that long circulating PEO-modified polymeric nanoparticles are efficient carriers for intracellular delivery of drugs and genes (15). For instance, PEO-PCL nanoparticles were found to efficiently encapsulate hydrophobic drugs, such as tamoxifen and paclitaxel, and deliver in tumor cells and in vivo (15,24). PCL was selected as a hydrophobic slow-eroding matrix that can provide sustained intracellular drug delivery. In addition, unlike other biodegradable polymers, such as poly(d,l-lactide-co-glycolide), the by-products of PCL degradation are not acidic and do not cause any toxicity in vivo.
Preparation and Characterization of PEO-PCL Nanoparticles
Particle Size and Surface Charge of the Control and Saquinavir-Containing Poly(Ethylene Oxide)-Modified Poly(Epsilon-Caprolactone) Nanoparticle Formulationsa
Particle size (nm)
Surface charge (mV)
High Resolution C1s Peak Analysis of ESCA on the Surface of Poly(Ethylene Oxide)-Modified Poly(Epsilon-Caprolactone) Nanoparticlesa
Relative C1s Peak Area (%)
PCL nanoparticles (in the absence of PF-108)
PCL-PF-108 with 5% (w/w) PF-108, as is
PCL-PF-108 with 5% (w/w) PF-108, washed once
PCL-PF-108 with 5% (w/w) PF-108, washed twice
PCL-PF-108 with 5% (w/w) PF-108, washed thrice
PCL-PF-108 with 15% (w/w) PF-108, as is
PCL-PF-108 with 15% (w/w) PF-108, washed once
PCL-PF-108 with 15% (w/w) PF-108, washed twice
PCL-PF-108 with 15% (w/w) PF-108, washed thrice
Saquinavir Incorporation and In Vitro Release Studies
Uptake and Distribution of PEO-PCL Nanoparticles in Macrophages
Intracellular Saquinavir Concentrations
Our study shows that an efficient nanoparticle formulation can be developed for saquinavir to ensure better encapsulation and release of the drug. A significant uptake of nanoparticles was also observed in the THP-1 cells of the monocyte/macrophage origin, which are known to shuttle the infected virions. The intracellular concentrations of saquinavir when administered in the nanoparticle formulations was significantly higher than from an aqueous solution. This strategy can potentially serve as a useful targeted drug delivery system for eradicating the viral sanctuaries in patients infected with HIV-1/AIDS.
The authors thank Professor Robert Campbell for the access to particle size and zeta potential instrument and Ms. Sushma Kommareddy for the SEM analysis. Additionally, Dr. Lara Gamble’s help with the ESCA investigations at the NESAC/BIO is gratefully acknowledged. NESAC/BIO is supported by the National Institutes of Health grant EB-002027.