The objective of this study is to engineer polylysine–heparin functionalized solid lipid nanoparticles (fSLNs) for the use of a vaginal microbicide delivery template for HIV prevention. The fSLNs are prepared using a modified phase-inversion technique followed by a layer-by-layer deposition method. The Box–Behnken experimental design is used to analyze the influence of three factors (X1 = bovine serum albumin concentration, X2 = pH of the aqueous phase, and X3 = lipid amount) on the particle mean diameter (PMD) measured by dynamic light scattering (DLS). Tenofovir is used as a model anti-HIV microbicide. The SLNs are also characterized for morphology, zeta potential (ζ), percent drug encapsulation efficiency (EE%), and cytotoxicity on a human vaginal epithelial cell line by electron microscopy, DLS, ultraviolet, and fluorescence spectroscopy, respectively. The statistical model predicts particle size (Y) with 90% confidence and the Y values are significantly affected by X1 and X2. The produced fSLNs appear noncytotoxic and exhibit a platelet-like shape with respective PMD, EE%, and ζ value of 153 nm, 8.3%, and −51mV. These fSLNs intended to be administered topically have the potential to enhance cellular uptake of hydrophobic microbicides and outdistance the virus during the HIV/AIDS infection process, possibly leading to more effective prevention of the disease transmission.
coating; colloids; formulation; functionalization; HIV prevention; lipids; nanoparticles; tenofovir; topical drug delivery
Low molecular weight heparin (LMWH) is the agent of choice for
anticoagulant therapy and prophylaxis of thrombosis and coronary syndromes.
However, its therapeutic use is limited due to poor oral bioavailability. The
aim of this study was to investigate the oral delivery of LMWH, ardeparin
formulated with 18-β glycyrrhetinic acid (GA), as an alternative to
currently used subcutaneous (sc) delivery. Drug transport through Caco-2 cell
monolayers was monitored in the presence and absence of GA by scintillation
counting and transepithelial electrical resistance. Regional permeability
studies using rat intestine were performed using a modified Ussing chamber. Cell
viability in the presence of various concentrations of enhancer was determined
by MTT assay. The absorption of ardeparin after oral administration in rats was
measured by an anti-factor Xa assay. Furthermore, the eventual mucosal
epithelial damage was histologically evaluated. Higher ardeparin permeability
(~7-fold) compared to control was observed in the presence of 0.02 %
GA. Regional permeability studies indicated predominant absorption in the
duodenal segment. Cell viability studies showed no significant cytotoxicity
below 0.01 % GA. Ardeparin oral bioavailability was significantly
increased (Frelative/S.C. = 13.3%)
without causing any damage to the intestinal tissues. GA enhanced the oral
absorption of ardeparin both in vitro and in vivo. The oral formulation of
ardeparin with GA could be absorbed in the intestine. These results suggest that
GA may be used as an absorption enhancer for the oral delivery of LMWH.
glycyrrhetinic acid; LMWH; Caco-2 cells; absorption enhancer; oral delivery
To develop spray dried mucoadhesive and pH-sensitive microspheres (MS) based on polymethacrylate salt intended for vaginal delivery of tenofovir (a model HIV microbicide) and assess their critical biological responses.
The formulation variables and process parameters are screened and optimized using a 24-1 fractional factorial design. The MS are characterized for size, zeta potential, yield, encapsulation efficiency, Carr’s index, drug loading, in vitro release, cytotoxicity, inflammatory responses and mucoadhesion.
The optimal MS formulation has an average size of 4.73 µm, Zeta potential of −26.3 mV, 68.9% yield, encapsulation efficiency of 88.7%, Carr’s index of 28.3 and drug loading of 2% (w/w). The MS formulation can release 90% of its payload in the presence of simulated human semen. At a concentration of 1 mg/ml, the MS are noncytotoxic to vaginal endocervical/epithelial cells and Lactobacillus crispatus when compared to control media. There is also no statistically significant level of inflammatory cytokine (IL1-α, IL-1β, IL-6, IL-8, and IP-10) release triggered by MS. The mucoadhesive property of MS formulation is 2-fold higher than that of 1% HEC gel formulation.
These data suggest the promise of using such MS as an alternative controlled microbicide delivery template by intravaginal route for HIV prevention.
pH-sensitive; mucoadhesive; tenofovir; spray drying; HIV/AIDS microbicide
Once metastatic, melanoma remains one of the most aggressive and morbid malignancies. Moreover, in past decades, the overall survival for advanced unresectable melanoma exhibited a constancy of poor prognosis. Low response rates and serious adverse effects have been characteristic of standard therapy based on a combination of chemotherapeutic agents or immunotherapy with IL-2. For example, the chemotherapy including dacarbazine, carmustin, cisplatin and tamoxifen is known as ‘Dartmouth regimen’ while the CVD regimen comprises carmustine, vinblastine and dacarbazine. Thus, there is an urgent and critical need to reformulate these bioactive agents using nanoscience and nanotechnology as alternative strategies. This article overviews current design and evaluation of nanomedicine undertaken to address this unmet medical need. The nanomedicines studied include polymeric nanoparticles, liposomes, polymersomes, dendrimers, cubosomes, niosomes and nanodiamonds. In this preclinical article, nanotechnology provides hope for effective treatment of this aggressive and largely treatment-resistant disease.
cancer; engineering; melanoma; nanomedicines; therapy
The purpose of this study was to develop and validate a rapid, sensitive, and specific reversed-phase high-performance liquid chromatography method for the quantitative determination of native tenofovir (TNF) for various applications. Different analytical performance parameters such as linearity, precision, accuracy, limit of quantification (LOQ), limit of detection (LOD), and robustness were determined according to International Conference on Harmonization (ICH) guidelines. A Bridge™ C18 column (150 × 4.6 mm, 5 μm) was used as stationary phase. The retention time of TNF was 1.54 ± 0.03 min (n = 6). The assay was linear over the concentration range of 0.1–10 μg/mL. The proposed method was sensitive with LOD and LOQ values equal to 50 and 100 ng/mL, respectively. The method was accurate with percent mean recovery from 95.41% to 102.90% and precise as percent RSD (relative standard deviation) values for intra-day, and inter-day precision were less than 2%. This method was utilized for the estimation of molar absorptivity of TNF at 259 nm (ε259 = 12,518 L/mol/cm), calculated from linear regression analysis. The method was applied for determination of percentage of encapsulation efficiency ( 22.93 ± 0.04%), drug loading (12.25 ± 1.03%), in vitro drug release profile in the presence of enzyme (43% release in the first 3 h) and purification analysis of hyaluronic acid-based nanomedicine.
high-performance liquid chromatography; hyaluronic acid; molar absorptivity; nanomedicine; tenofovir
This study is designed to test the hypothesis that Tenofovir(TNF)ortenofovir disoproxil fumarate (TDF) loaded nanoparticles (NPs)prepared with a blend of poly(lactic-co-glycolic acid) (PLGA) and methacrylic acid copolymer (Eudragit® S-100, or S-100)are noncytotoxic and exhibit significant pH-responsive release of anti-HIV microbicides in presence of human semen. After NPs preparation by emulsification diffusion, their size, encapsulation efficiency (EE%), drug release profile, morphology, and cytotoxicity are characterized by dynamic light scattering, spectrophotometry, transmission electron microscopy, and cellular viability assay/transepithelial electrical resistance measurement, respectively. Cellular uptake was elucidated by fluorescence spectroscopy and confocal microscopy. The NP shavean average size of 250 nm, maximal EE% of 16.1% and 37.2% for TNF and TDF, respectively. There is a 4-fold increase in the drug release rate from 75% S-100 blendin the presence of semen fluid simulant over 72 hr. At a concentration up to 10 mg/ml, the PLGA/S-100 NPs are noncytotoxic for 48 hr to vaginal endocervical/epithelial cells and Lactobacillus crispatus. The particle uptake (~50% in 24hr.) by these vaginal cell lines mostly occurred through caveolin-mediated pathway. These data suggest the promise of using PLGA/S-100 NP as an alternative controlled drug delivery system in intravaginal delivery of an anti-HIV/AIDS microbicide.
pH-responsive; nanoparticles; tenofovir; tenofovir disoproxil fumarate; topical delivery; HIV/AIDS microbicide
The objective of this study was to engineer a model anti-HIV microbicide (Tenofovir) loaded chitosan based nanoparticles (NPs). Box-Behnken design allowed to assess the influence of formulation variables on the size of NPs and drug encapsulation efficiency (EE%) that were analyzed by dynamic light scattering and UV spectroscopy, respectively. The effect of the NPs on vaginal epithelial cells and Lactobacillus crispatus viability and their mucoadhesion to porcine vaginal tissue were assessed by cytotoxicity assays and fluorimetry, respectively. In the optimal aqueous conditions, the EE% and NPs size was 5.83% and 207.97nm, respectively. With 50% (v/v) ethanol/water as alternative solvent, these two responses increased to 20% and 602 nm, respectively. Drug release from medium (281 nm) and large size (602 nm)-sized NPs fitted the Higuchi (r2=0.991) and first-order release (r2=0.999) models, respectively. These NPs were not cytotoxic to both the vaginal epithelial cell line and Lactobacillus for 48 hours. When the diameter of the NPs decreased from 900 nm to 188 nm, the mucoadhesion increased from 6% to 12%. However, the combinatorial effect of EE% × mucoadhesion for larger size NPs was the highest. Overall, large-size, microbicide loaded chitosan NPs appeared to be promising nanomedicines for the prevention of HIV transmission.
Chitosan nanoparticles; HIV prevention; Mucoadhesion; Tenofovir; Vaginal drug delivery
This study is designed to test the hypothesis that docetaxel [Doc] containing oily core nanocapsules [NCs] could be successfully prepared with a high percentage encapsulation efficiency [EE%] and high drug loading. The oily core NCs were generated according to the emulsion solvent diffusion method using neutral Labrafac CC and poly(d, l-lactide) [PLA] as oily core and shell, respectively. The engineered NCs were characterized for particle mean diameter, zeta potential, EE%, drug release kinetics, morphology, crystallinity, and cytotoxicity on the SUM 225 breast cancer cell line by dynamic light scattering, high performance liquid chromatography, electron microscopies, powder X-ray diffraction, and lactate dehydrogenase bioassay. Typically, the formation of Doc-loaded, oily core, polyester-based NCs was evidenced by spherical nanometric particles (115 to 582 nm) with a low polydispersity index (< 0.05), high EE% (65% to 93%), high drug loading (up to 68.3%), and a smooth surface. Powder X-ray diffraction analysis revealed that Doc was not present in a crystalline state because it was dissolved within the NCs' oily core and the PLA shell. The drug/polymer interaction has been indeed thermodynamically explained using the Flory-Huggins interaction parameters. Doc release kinetic data over 144 h fitted very well with the Higuchi model (R2 > 0.93), indicating that drug release occurred mainly by controlled diffusion. At the highest drug concentration (5 μM), the Doc-loaded oily core NCs (as a reservoir nanosystem) enhanced the native drug cytotoxicity. These data suggest that the oily core NCs are promising templates for controlled delivery of poorly water soluble chemotherapeutic agents, such as Doc.
docetaxel; polylactide; emulsion diffusion; nanocapsules; drug loading
The purpose of this work was to optimize the process parameters required for the production of spray-dried oily core nanocapsules (NCs) with targeted size and drug yield using a two-level four-factor fractional factorial experimental design (FFED). The coded process parameters chosen were inlet temperature (X1), feed flow rate (X2), atomizing air flow (X3), and aspiration rate (X4). The produced NCs were characterized for size, yield, morphology, and powder flowability by dynamic light scattering, electron microscope, Carr’s index, and Hausner ratio measurement, respectively. The mean size of produced NCs ranged from 129.5 to 444.8 nm, with yield varying from 14.1% to 31.1%. The statistical analysis indicated an adequate model fit in predicting the effect of process parameters affecting yield. Predicted condition for maximum yield was: inlet temperature 140°C, atomizing air flow 600 L/h, feed flow rate 0.18 L/h, and aspiration air flow set at 100%, which led to a yield of 30.8%. The morphological analysis showed the existence of oily core and spherical nanostructure. The results from powder flowability analysis indicated average Carr’s index and Hausner ratio of 42.77% and 1.76, respectively. Spray-dried oily core NCs with size lower than 200 nm were successfully produced, and the FFED proved to be an effective approach in predicting the production of spray-dried NCs of targeted yield.
fractional factorial design; indomethacin; oily core nanocapsules; process variables; spray drying
The purpose of this study was to investigate the physicochemical properties of dacarbazine-loaded cubosomes. The drug-loaded cubosome nanocarriers were prepared by a fragmentation method and then freeze dried. They were then characterized for size, morphology, thermal behavior, and crystallography using dynamic light scattering, transmission electron microscopy (TEM), differential scanning calorimetry (DSC), and powder X-ray diffraction (PXRD), respectively. The drug loading and encapsulation efficiency were determined by UV spectrophotometry. The results showed that the prepared dacarbazine-loaded cubosomes had mean diameters ranging from 86 to 106 nm. In addition to the TEM, the characteristic peaks from PXRD data suggested that the freeze-dried nanoformulations were indeed cubic in nature. DSC and PXRD analysis suggested the 0.06 or 0.28% w/w actual drug loaded inside cubosomes was in the amorphous or molecular state. These physicochemical characteristics would affect the nanoformulation shelf-life, efficacy, and safety.
cubosome; dacarbazine; thermal analysis; X-ray diffraction
The current advances in chronobiology and the knowledge gained from chronotherapy of selected diseases strongly suggest that “the one size fits all at all times” approach to drug delivery is no longer substantiated, at least for selected bioactive agents and disease therapy or prevention. Thus, there is a critical and urgent need for chronopharmaceutical research (e.g., design and evaluation of robust, spatially and temporally controlled drug delivery systems that would be clinically intended for chronotherapy by different routes of administration). This review provides a brief overview of current delivery system intended for chronotherapy. In theory, such an ideal “magic pill” preferably with affordable cost, would improve the safety, efficacy and patient compliance of old and new drugs. However, currently, there are three major hurdles for the successful transition of such system from laboratory to patient bedside. These include the challenges to identify adequate (i) rhythmic biomaterials and systems, (ii) rhythm engineering modeling, perhaps using system biology and (iii) regulatory guidance.
Chronopharmaceutics; delivery systems; chronotherapy; rhythmic biomaterials; modeling; regulations
The purpose of this study is to investigate the combined influence of process parameters (independent variables) such as homogenization speed (X1), duration (X2), and temperature (X3) during the preparation of dacarbazine-loaded cubosomes. Box–Behnken design was used to rationalize the influence of these three factors on two responses, namely particle size (Y1) and encapsulation efficiency (Y2). Independent and dependent variables were analyzed with multiple regressions to establish a full-model second-order polynomial equation. F value was calculated to confirm the omission of insignificant parameters or interactions of parameters from the analysis to derive a reduced-model polynomial equation to predict the Y1 and Y2 of dacarbazine-loaded cubosomes. Pareto charts were also obtained to show the effects of X1, X2, and X3 on Y1 and Y2. For Y1, there was a model validated for more accurate prediction of response parameter by performing checkpoint analysis. The optimization process and Pareto charts were obtained automatically and they predicted the levels of independent parameters X1, X2, and X3 (0.889794, 0.11886, and 0.56201, respectively) and minimized Y1. The optimal process parameters (homogenization’s speed = ~24,000 rpm, duration = 5.5 min, and temperature = 76°C) led to the production of cubosomes with 85.6 nm in size and 16.7% in encapsulation efficiency. The Box–Behnken design proved to be a useful tool in the preparation and optimization of dacarbazine-loaded cubosomes. For encapsulation efficiency (Y2), further studies are needed to enhance the result and improve the model for such water-soluble drug encapsulation in cubosomes.
Box–Behnken design; cubosomes; dacarbazine; optimization; process
The purpose of this study was to investigate the combined influence of three-level, three-factor variables on the formulation of dacarbazine (a water-soluble drug) loaded cubosomes. Box–Behnken design was used to obtain a second-order polynomial equation with interaction terms to predict response values. In this study, the selected and coded variables X1, X2, and X3 representing the amount of monoolein, polymer, and drug as the independent variables, respectively. Fifteen runs of experiments were conducted, and the particle size (Y1) and encapsulation efficiency (Y2) were evaluated as dependent variables. We performed multiple regression to establish a full-model second-order polynomial equation relating independent and dependent variables. A second-order polynomial regression model was constructed for Y1 and confirmed by performing checkpoint analysis. The optimization process and Pareto charts were obtained automatically, and they predicted the levels of independent coded variables X1, X2, and X3 (−1, 0.53485, and −1, respectively) and minimized Y1 while maximizing Y2. These corresponded to a cubosome formulation made from 100 mg of monoolein, 107 mg of polymer, and 2 mg with average diameter of 104.7 nm and an encapsulation efficiency of 6.9%. The Box–Behnken design proved to be a useful tool to optimize the particle size of these drug-loaded cubosomes. For encapsulation efficiency (Y2), further studies are needed to identify appropriate regression model.
Box–Behnken design; cubosomes; dacarbazine; formulation variables
The development of a non-invasive drug delivery system for unfractionated heparin (UFH) and low molecular weight heparins (LMWHs) has been the elusive goal of several research groups since the initial discovery of this glycosaminogylcan by McLean in 1916. After a brief update on current parenteral formulations of UFH and LMWHs, this review revisits past and current strategies intended to identify alternative routes of administration (e.g. oral, sublingual, rectal, nasal, pulmonary and transdermal). The following strategies have been used to improve the bioavailability of this bioactive macromolecule by various routes: (i) enhancement in cell-membrane permeabilization, (ii) modification of the tight-junctions, (iii) increase in lipophilicity and (iv) protection against acidic pH of the stomach. Regardless of the route of administration, a simplified unifying principle for successful non-invasive macromolecular drug delivery may be: “to reversibly overcome the biological, biophysical and biochemical barriers and to safely and efficiently improve the in vivo spatial and temporal control of the drug in order to achieve a clinically acceptable therapeutic advantage”. Future macromolecular drug delivery research should embrace a more systemic approach taking into account recent advances in genomics/proteomics and nanotechnology.
Alternative routes; Bioactive macromolecule; Drug delivery; Non-invasive; Heparin
Zonula occludens toxin (Zot) is an enterotoxin obtained from the
bacterium vibrio cholerae that has been shown to reversibly and
safely open the tight junctions and enhance paracellular transport. AT1002 is a
novel synthetic hexapeptide derived from Zot. The hypothesis to be tested in
this study is that AT1002 enhances the oral absorption of ardeparin, a low
molecular weight heparin (LMWH). To test this hypothesis, drug transport through
Caco-2 cell monolayers was monitored in the presence and absence of AT1002.
Regional permeability studies using rat intestine were performed. Cell viability
in the presence of various concentrations of enhancer was determined. The
absorption of ardeparin after oral administration in rats was measured by
anti-factor Xa assay. Furthermore, the eventual mucosal and epithelial damage
was histologically evaluated. Higher ardeparin permeability (~2-fold) compared
to control was observed in the presence of 0.025% of AT1002.
Regional permeability studies revealed that the permeability of ardeparin across
the duodenal membrane was improved by the AT1002. Cell viability studies showed
no significant cytotoxicity below 0.0028% of AT1002. In the presence
of 100 μg/kg of AT1002, ardeparin oral bioavailability was
significantly increased (Frelative/s.c ~
20.5%). Furthermore, AT1002 at a dose of 100 μg/kg did
not induce any observable morphological damage on gastrointestinal (GI) tissues
in vivo. These in vivo and in
vitro results suggest that the co-administration of LMWH with
AT1002 may be a useful delivery strategy to increase its permeability and hence
AT1002; low molecular weight heparin; enhancer; oral delivery; zonula occludens toxin
The primary objective of this study was to evaluate sodium caprate as an
oral penetration enhancer for low molecular weight heparin (LMWH), ardeparin.
In vitro studies using Caco-2 cell monolayer indicated that
0.0625% of sodium caprate gave approximately 2-fold enhancement of
ardeparin compared to negative control with almost 100% cell
survival as evaluated by MTT cytotoxicity assay. In vivo
studies in rats with ardeparin (1200 IU/kg) and sodium caprate (100 mg/kg) led
to a relative bioavailability of 27% with plasma anti-factor Xa
levels within the therapeutic range (> 0.2 IU/ml). Moreover, under these
conditions, histological examination provided evidence that there was no damage
to the gastrointestinal wall. Regional permeability studies using rat intestine
indicated the colon as the region of maximum permeation. These results suggest
that, at the dose administered, sodium caprate acts as a relatively safe and
efficient absorption enhancer in the quest for alternatives for the oral
delivery of LMWH.
Sodium caprate; low molecular weight heparin; Caco-2 cell monolayer; absorption enhancer; ardeparin; oral absorption
Sucrose esters (SE) are surfactants with potential pharmaceutical applications because of their low toxicity, biocompatibility, and excellent biodegradability. The objective of the study was to investigate SE as alternative surfactants in stabilizing emulsions for the preparation of protein-loaded microparticles. To achieve this goal, using bovine serum albumin as model protein and 75/25 poly(d,1-lactide-co-glycolide) as polymer carrier, we have investigated the influence of the following formulation variables on particle characteristics: (1) SE concentration from 0.01% to 1% (wt/vol), (2) hydrophile-lipophile balance (HLB) value of SE from 6 to 15, and (3) the nature of emulsion stabilizer. The formulations were characterized using ATR-FTIR spectroscopy, bicinchoninic acid protein assay, optical microscopy and SDS-PAGE. Results showed that at 0.05% (wt/vol) surfactant concentration, SE with HLB of 6 to 15 provided discrete and spherical microparticles with the highest encapsulation efficiency compared with controls polyvinyl alcohol (PVA) and poloxamer 188. These results may be explained by the difference in critical micelle concentration, diffusion, and partition coefficient among the tested surfactants. HLB values were consistent with SE spectral data. The protein molecular weight was preserved after the encapsulation process. The effective SE concentration was far less (20-to 200-fold) than that is usually required for PVA in microencapsulation of proteins. However, the encapsulation efficiency was relatively lower (∼13.5%). These preliminary results suggest that it may be desirable to optimize such formulations in vitro and in vivo for SE to be eventually used as altermative surfactants in the development of microparticulate systems for parenteral delivery of protein and gene medicines.
proteins; microencapsulation; solvent evaporation; sucrose ester; surfactants