Multifunctional graphene-based magnetic nanocarriers for combined hyperthermia and dual stimuli-responsive drug delivery.

The synthesis of hydrophilic graphene-based yolk-shell magnetic nanoparticles functionalized with copolymer pluronic F-127 (GYSMNP@PF127) is herein reported to achieve an efficient multifunctional biomedical system for mild hyperthermia and stimuli-responsive drug delivery. In vitro tests revealed the extraordinary ability of GYSMNP@PF127 to act as smart stimuli-responsive multifunctional nanomedicine platform for cancer therapy, exhibiting (i) an outstanding loading capacity of 91% (w/w, representing 910 µg mg-1) of the chemotherapeutic drug doxorubicin, (ii) a high heating efficiency under an alternating (AC) magnetic field (intrinsic power loss ranging from 2.1-2.7 nHm2 kg-1), and (iii) a dual pH and thermal stimuli-responsive drug controlled release (46% at acidic tumour pH vs 7% at physiological pH) under AC magnetic field, in just 30 min. Additionally, GYSMNP@PF127 presents optimal hydrodynamic diameter (DH = 180 nm) with negative surface charge, high haemocompatibility for blood stream applications and tumour cellular uptake of drug nanocarriers. Due to its physicochemical, magnetic and biocompatibility properties, the developed graphene-based magnetic nanocarrier shows high promise as dual exogenous (AC field)/endogenous (pH) stimuli-responsive actuators for targeted thermo-chemotherapy, combining magnetic hyperthermia and controlled drug release triggered by the abnormal tumour environment. The presented strategy and findings can represent a new way to design and develop highly stable added-value graphene-based nanostructures for the combined treatment of cancer.

Nanoemulsion containing 8-methoxypsoralen for topical treatment of dermatoses: Development, characterization and ex vivo permeation in porcine skin.

Oral therapy with 8-methoxypsoralen (8-MOP) may cause major side effects, whereas the topical treatment might not be much effective due to the low penetration induced by typical formulations. Therefore, the objectives of this work are the development and characterization of a nanoemulsion (NE) containing 8-MOP together with an ex vivo permeation study, monitored by a validated HPLC-Fluo method, to determine the amount of drug retained in viable skin (epidermis (E) and dermis (D)) and in stratum corneum (SC). The optimized conditions for NE formulation were achieved by full factorial designs (25 and 32): 60 s and 60% of ultrasound time and potency, respectively; 10 mL of final volume; 2% v/v of oil phase (clove essential oil); and 10% m/v of Poloxamer 407. The NE showed mean droplet diameter of 24.98 ±â€¯0.49 nm, polydispersity index (PDI) of 0.091 ±â€¯0.23, pH values of 6.54 ±â€¯0.06, refractive index of 1.3525 ±â€¯0.0001 and apparent viscosity of 51.15 ±â€¯3.66 mPa at 20 °C. Droplets with nanospherical diameters were also observed by transmission electron microscopy (TEM). Ex vivo permeation study showed that 8.5% of the applied 8-MOP dose permeated through the biological membranes, with flux (J) of 1.35 µg cm-2 h-1. The drug retention in E + D and in SC was 10.15 ±â€¯1.36 and 1.95 ±â€¯0.71 µg cm-2, respectively. Retention in viable skin induced by the NE was almost two-fold higher than a compounded cream (5.04 ±â€¯0.30 µg cm-2). These results suggested that the developed NE is a promising alternative for 8-MOP topical therapy when compared to commercial formulations.

Enhanced oral bioavailability of an antipsychotic drug through nanostructured lipid carriers.

Olanzapine is an atypical antipsychotic, undergoes extensive first pass metabolism, also has poor aqueous solubility and belongs to BCS (Biopharmaceutical Classification System) Class II drug) exhibit low oral bioavailability. To overcome this and to enhance the bioavailability, intestinal lymphatic transport of drugs can be exploited through Nano structured lipid carriers (NLCs). The NLCs were formulated by solvent diffusion method using solid lipid (glyceryl tripalmitate), liquid lipid (castor oil) and surfactants (Pluronic F-68, Soylecithin). The formulated NLCs were characterized for physico-chemical properties, in-vitro release studies and in-vivo oral bioavailability. F6 has shown average particle size of 158.5 nm with PI of 0.115 indicating narrow particle size distribution and follows uni modal distribution. It was found that the batch with stearyl amine has a zeta potential of 28.39 mV which confers stability to the dispersion. Bioavailability studies indicate that there was more than 5½-fold increase in oral bioavailability in case of NLCs (F6) compared to olanzapine suspension which indicates that NLCs provided sustained release of the drugs, and these systems can be the preferred as drug carriers for lipophilic drugs in long term disease conditions such as schizophrenia for enhanced bioavailability.

Development and in vivo evaluation of an innovative "Hydrochlorothiazide-in Cyclodextrins-in Solid Lipid Nanoparticles" formulation with sustained release and enhanced oral bioavailability for potential hypertension treatment in pediatrics.

An innovative pediatric oral formulation of hydrochlorothiazide (HCT) (2mg/mL), endowed with improved bioavailability and sustained release properties and suitable for the hypertension treatment in pediatric patients, was developed by combining the drug-cyclodextrin complexation and the incorporation of the complex into Solid Lipid Nanoparticles (SLN). Precirol®ATO5-based SLN, with two different surfactants (Pluronic®F68 and Tween®80) loaded with the drug as such or as binary system with hydroxypropyl-beta-cyclodextrin (HPßCd) and sulfobutyl-ether-beta-cyclodextrin (SBEßCd) both as physical mixture (P.M.) or coground product (GR), were prepared using the hot high-shear homogenization followed by ultrasonication method. Loading of the drug:HPßCd both as P.M. and GR gave rise to nanoparticle formation, differently from the HCT:SBEßCd ones, with an entrapment efficiency of about 65%. Such SLN formulations showed an improvement of the drug release rate compared both to the drug suspension and to the free drug-loaded SLN. In all cases the SLN containing the GR systems exhibited better performances than the corresponding with P.M. However, the presence of Tween®80 gave rise to the complete drug release after only 150min, without providing a sustained release, whereas Pluronic®F68-based SLN containing GR were able to assure a sustained release over the time achieving more than 75% drug released at the end of the test, maintaining a constant 1.8-fold increase respect to simple drug suspension. Pluronic®F68-based SLN showed a pharmaceutically acceptable stability up to three months. In vivo studies highlighted the effectiveness of such formulations, enabling a concomitant increased diuretic effect and a sustained drug release and, consequently, enhanced HCT oral bioavailability.

Multifunctional theranostic Pluronic mixed micelles improve targeted photoactivity of Verteporfin in cancer cells.

Nanotechnology development provides new strategies to treat cancer by integration of different treatment modalities in a single multifunctional nanoparticle. In this scenario, we applied the multifunctional Pluronic P123/F127 mixed micelles for Verteporfin-mediated photodynamic therapy in PC3 and MCF-7 cancer cells. Micelles functionalization aimed the targeted delivery by the insertion of biotin moiety on micelle surface and fluorescence image-based through rhodamine-B dye conjugation in the polymer chains. Multifunctional Pluronics formed spherical nanoparticulated micelles that efficiently encapsulated the photosensitizer Verteporfin maintaining its favorable photophysical properties. Lyophilized formulations were stable at least for 6months and readily reconstituted in aqueous media. The multifunctional micelles were stable in protein-rich media due to the dual Pluronic mixed micelles characteristic: high drug loading capacity provided by its micellar core and high kinetic stability due its biocompatible shell. Biotin surface functionalized micelles showed higher internalization rates due biotin-mediated endocytosis, as demonstrated by competitive cellular uptake studies. Rhodamine B-tagged micelles allowed monitoring cellular uptake and intracellular distribution of the formulations. Confocal microscopy studies demonstrated a larger intracellular distribution of the formulation and photosensitizer, which could drive Verteporfin to act on multiple cell sites. Formulations were not toxic in the dark condition, but showed high Verteporfin-induced phototoxicity against both cancer cell lines at low drug and light doses. These results point Verteporfin-loaded multifunctional micelles as a promising tool to further developments in photodynamic therapy of cancer.

Preparation, properties and anticancer effects of mixed As4S4/ZnS nanoparticles capped by Poloxamer 407.

Arsenic sulfide compounds have a long history of application in a traditional medicine. In recent years, realgar has been studied as a promising drug in cancer treatment. In this study, the arsenic sulfide (As4S4) nanoparticles combined with zinc sulfide (ZnS) ones in different molar ratio have been prepared by a simple mechanochemical route in a planetary mill. The successful synthesis and structural properties were confirmed and followed via X-ray diffraction and high-resolution transmission electron microscopy measurements. The morphology of the particles was studied via scanning electron microscopy and transmission electron microscopy methods and the presence of nanocrystallites was verified. For biological tests, the prepared As4S4/ZnS nanoparticles were further milled in a circulation mill in a water solution of Poloxamer 407 (0.5wt%), in order to cover the particles with this biocompatible copolymer and to obtain stable nanosuspensions with unimodal distribution. The average size of the particles in the nanosuspensions (~120nm) was determined by photon cross-correlation spectroscopy method. Stability of the nanosuspensions was determined via particle size distribution and zeta potential measurements, confirming no physico-chemical changes for several months. Interestingly, with the increasing amount of ZnS in the sample, the stability was improved. The anti-cancer effects were tested on two melanoma cell lines, A375 and Bowes, with promising results, confirming increased efficiency of the samples containing both As4S4 and ZnS nanocrystals.

Poloxamer 407/188 binary thermosensitive hydrogels as delivery systems for infiltrative local anesthesia: Physico-chemical characterization and pharmacological evaluation.

In this study, we reported the development and the physico-chemical characterization of poloxamer 407 (PL407) and poloxamer 188 (PL188) binary systems as hydrogels for delivering ropivacaine (RVC), as drug model, and investigate their use in infiltrative local anesthesia for applications on the treatment of post-operative pain. We studied drug-micelle interaction and micellization process by light scattering and differential scanning calorimetry (DSC), the sol-gel transition and hydrogel supramolecular structure by small-angle-X-ray scattering (SAXS) and morphological evaluation by Scanning Electron Microscopy (SEM). In addition, we have presented the investigation of drug release mechanisms, in vitro/in vivo toxic and analgesic effects. Micellar dimensions evaluation showed the formation of PL407-PL188 mixed micelles and the drug incorporation, as well as the DSC studies showed increased enthalpy values for micelles formation after addition of PL 188 and RVC, indicating changes on self-assembly and the mixed micelles formation evoked by drug incorporation. SAXS studies revealed that the phase organization in hexagonal structure was not affected by RVC insertion into the hydrogels, maintaining their supramolecular structure. SEM analysis showed similar patterns after RVC addition. The RVC release followed the Higuchi model, modulated by the PL final concentration and the insertion of PL 188 into the system. Furthermore, the association PL407-PL188 induced lower in vitro cytotoxic effects, increased the duration of analgesia, in a single-dose model study, without evoking in vivo inflammation signs after local injection.

Preparation of Pluronic Grafted Dendritic alpha,epsilon-poly(L-lysine)s and Characterization as a Delivery Adjuvant of Antisense Oligonucleotide.

A series of pluronic grafted dendritic alpha,epsilon-poly(L-lysine)s (DPL-PF127) were synthesized by a conjugation reaction and evaluated the potential use of DPL-PF127 as a delivery agent of antisense oligonucleotide into A375 B3 cells. The structural features of the DPL-PF127 were identified by NMR and FT-IR. The number of pluronic F127 on DPL surface, determined by fluorescamine assay, increased proportionally to the mole ratio between DPL and activated PF127 in reaction. DPL- PF127 showed the physical properties of decrease in zetapotential and increase in size as the mole ratio of PF127 to DPL increased. The complex formation of DPL-PF127 with oligonucleotide was confirmed by running capillary zone electrophoresis (CZE) and agarose gel electrophoresis. DPL-PF127, prepared at the mole ratio of 1:10 in reaction, was the most suitable as a delivery adjuvant of oligonucleotide. In addition, DPL-PF127/oligonucleotide complexes were taken into A375B3 cell without cellular toxicity and delivered antisense oligonucleotide into cell.

Physicochemical, pharmacokinetic and pharmacodynamic evaluations of novel ternary solid dispersion of rebamipide with poloxamer 407.

This study was conducted primarily to improve the solubility of rebamipide, a poorly water-soluble anti-ulcer drug, using novel ternary solid dispersion (SD) systems and secondly to evaluate the effect of solubility enhancement on its pharmacokinetic (PK) and pharmacodynamic (PD) profile. After dissolving the three components in aqueous medium, ternary SD containing the drug, sodium hydroxide (NaOH) and PVP-VA 64 was achieved by spray drying method, which was used as primary SD. Poloxamer 407, a surfactant polymer, was incorporated in this primary SD by four different methods: co-grinding, physical mixing, melting or spray drying. SD was then characterized by dissolution test, differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD) and Fourier transform infrared spectroscopy (FT-IR). The spray dried SD of poloxamer 407 together with primary SD displayed highest dissolution rate of the drug of about 70% after 2 h. DSC, PXRD and FT-IR characterized the amorphous state and molecular dispersion of the drug in the SD. PK and PD studies in Sprague-Dawley rats revealed that the bioavailability of the drug using optimal SD was about twofold higher than that of reference product, and the irritation area of stomach was significantly reduced in the ulcer-induced rat model using optimal SD as compared to the reference product.

I. Poloxamer-formulated plasmid DNA-based human cytomegalovirus vaccine: evaluation of plasmid DNA biodistribution/persistence and integration.

Preclinical studies were conducted in mice and rabbits to evaluate biodistribution/persistence and potential integration of plasmid DNA (pDNA) after intramuscular administration of a poloxamer-formulated pDNAbased vaccine, VCL-CT01, encoding gB, pp65, and IE1 human cytomegalovirus (hCMV) immunogens. Tissue distribution in mice vaccinated with VCL-CT01 was compared with that in mice vaccinated with a phosphate- buffered saline (PBS)-formulated control pDNA vaccine. Residual pDNA copy number (PCN), in selected tissues collected on days 3, 30, and 60 after vaccination, was measured by quantitative polymerase chain reaction. In VCL-CT01-vaccinated mice and in control pDNA-vaccinated mice, pDNA was below the limit of detection by day 60 in all tissues except the injection site. Clearance of pDNA from the injection site was slower in VCL-CT01-vaccinated mice compared with PBS-pDNA-vaccinated mice. An integration study was conducted in rabbits to determine whether pDNA integration into the genome of the vaccinated animal contributed to pDNA persistence. Residual pDNA in VCL-CT01-injected rabbit muscle collected 60 days after vaccination (geometric mean of 1085 PCN/microg total DNA) was comparable to that observed in VCL-CT01- injected mouse muscle (geometric mean of 1471 PCN/microg total DNA) collected at the same time point. pDNA integration was not detectable by column agarose gel electrophoresis despite the persistence of pDNA at the injection site 60 days after vaccination. Therefore the risk of genomic integration of hCMV pDNA formulated with poloxamer was considered negligible.

Preparation and evaluation of a chitosan salt-poloxamer 407 based matrix for buccal drug delivery.

The aim of this work was to prepare and evaluate a matrix for buccal drug delivery composed of a chitosan salt and poloxamer 407. Different chitosan salts were formed by reacting chitosan with acetic, citric, and lactic acid. Various proportions of poloxamer 407 were added to the aqueous solution of chitosan salt, and the residue obtained by lyophilisation was compressed into discs, using a 30 kN compression force. An experimental design (3(2)) was used to study the influence of the type of chitosan salt and of the relative amount of poloxamer on drug release capacity, swelling, erosion, and mucoadhesiveness of matrices. The results showed that matrix properties depended significantly on both relative amount of poloxamer and chitosan salt type. The rank orders of chitosan salts for the four processes evaluated were as follows: drug release: chitosan acetate>chitosan citrate>chitosan lactate; swelling: chitosan lactate>chitosan acetate=chitosan citrate; erosion: chitosan citrate>chitosan lactate>chitosan acetate; mucoadhesion: chitosan lactate>chitosan acetate=chitosan citrate. Mucoadhesion was particularly favoured when poloxamer 407 was present at about 30% (w/w). The matrix composed of chitosan lactate and poloxamer 407 showed the best characteristics for buccal administration.

The effects of emulsifying agents on disposition of lipid-soluble drugs included in fat emulsion.

The uses for drug delivery systems of two soybean oil fat emulsions prepared with an emulsifying agent, phosphatidyl choline (PC) or Pluronic F-127 (PLU), were examined comparatively in vivo and in vitro. In the presence of lipoprotein lipase (LPL) in vitro, the mean particle size of the PLU emulsion changed less than that of the PC emulsion. The production of non-esterified fatty acid (NEFA) from the PLU emulsion in the presence of LPL was smaller than that from the PC emulsion. These in vitro results indicate that the PLU emulsion is more stable than the PC emulsion. Plasma NEFA concentration following intravenous administration of the emulsions decreased with time for the PC emulsion, but was kept lower and constant for the PLU emulsion, supporting the in vitro stability data. The order of plasma cyclosporine A (CsA) concentration following intravenous administration in the above two emulsions and the mixed solution of polyethylene glycol 400 (PEG) and dimethylamide (DMA) in rats was PLU emulsion>PC emulsion>PEG/DMA solution. The plasma concentration was maintained higher and tissue distribution lower for the PLU emulsion than for other formulations. The uptake of oil violet (OV) into the rat parenchymal cells from the PLU emulsion was approximately half that from the PC emulsion, but the uptake into the Kupffer cells was almost equal in both emulsions. In conclusion, these emulsifying agents can control plasma elimination and tissue distribution of lipophilic drugs included in the emulsion. The use of the emulsion formulation makes it possible to avoid side effects through the reduction of drug uptake into non-targeted tissues.

Enhancing the bioavailability of ABT-963 using solid dispersion containing Pluronic F-68.

Solid dispersions using Pluronic F-68 as a carrier were studied for improving the dissolution and bioavailability of ABT-963, a poorly water-soluble compound. The solid dispersions were prepared either by evaporation of the ethanol solutions containing ABT-963 and Pluronic, or by cooling the hot melt of the drug in the carrier. The dispersions were characterized using differential scanning calorimetry, powder X-ray diffractometry, scanning electron microscopy, elemental mapping, and by constructing the melting point phase diagram. In vitro dissolution and in vivo oral bioavailability in fasted dogs were compared for the solid dispersion and a conventional IR capsule formulation. Results showed that, at a composition of approximately 7.5%, ABT-963 formed a eutectic mixture with Pluronic F-68. Both the drug and the polymer were crystalline in the solid dispersion with a wide range of composition of each component. The solid dispersion substantially increased the in vitro dissolution rate of ABT-963. Dosing of the dispersion to fasted dogs resulted in a significant increase of oral bioavailability compared with the conventional IR capsule formulation. These results show that solid dispersion is a promising approach for developing ABT-963 drug products.

Formulation and preliminary in vivo dog studies of a novel drug delivery system for the treatment of periodontitis.

A novel drug delivery system for the treatment of periodontitis was developed using two components. The first was tetracycline base loaded into the microtubular excipient halloysite, which was coated with chitosan to further retard drug release. Encapsulation efficiencies of 32.5% were achieved with the loading procedure, with tetracycline base showing in vitro release for up to 50 days in simulated gingival crevicular fluid. The second component developed was a vehicle for the drug loaded coated halloysite, which was primarily based on the thermoresponsive polymer, poloxamer 407. A concentration of 20% was chosen with the thermoresponsivity of the system modified using PEG 20,000 so that the mobile product at room temperature would gel by temperature rise following syringing into a periodontal pocket. Retention of the overall system in the pocket was further improved by the addition of octyl cyanoacrylate (OCA). The thermoresponsivity of the poloxamer 407 system proved to be sensitive to the presence of added excipients with the levels of PEG 20,000 and OCA requiring modification in the presence of the halloysite component. A final formulation was developed which consisted of 200 mg of halloysite double loaded with tetracycline base and coated with chitosan, suspended in 1 ml of poloxamer 407 20% (w/w), PEG 20,000 0.5% (w/w), OCA 1.0% (w/w), water to 100%, adjusted to pH 4. The syringeability of this formulation at various temperatures was evaluated to ensure ease of delivery to the periodontal pocket. A stability study was performed to examine the change in thermoresponsivity over time, with the final formulation found to be stable for at least 9 months when stored at room temperature (approximately 20 degrees C). This formulation offered ease of delivery to the periodontal pocket and sustained release of the antibiotic for up to 6 weeks. The formulation had preliminary in vivo testing performed in dogs to determine levels of drug release, antimicrobial activity and retentive ability of the product. A wound pocket creation model was developed for the purposes of the trial. The product was easy to deliver to the pockets with application times of less than 1 min. Results showed the product was retained in the pocket for up to 6 weeks with effective tetracycline levels released locally over this time period, which achieved good antibacterial activity.

Development and in-vitro evaluation of sustained release poloxamer 407 (P407) gel formulations of ceftiofur.

The objective of this study was to develop sustained release Poloxamer 407 (P407) gel formulations of ceftiofur for treating foot infections in cattle. The formulations contained 25-35% (w/v) P407 alone or with polyvinyl pyrrolidone (PVP), carboxy methylcellulose (CMC), or hydroxylpropyl methylcellulose (HPMC) as an additive. The in-vitro release profiles of ceftiofur from the P407 formulations and the gel dissolution profiles were obtained simultaneously. Ceftiofur release followed zero order kinetics and correlated well with the weight percentage of P407 dissolved, indicating that the overall rate of release of ceftiofur is controlled by dissolution of the P407. An increase in P407 content from 25 to 35% resulted in a decrease in the rate of ceftiofur release. However, it appears that other factors may have also affected the drug release rate. Inclusion of PVP, CMC, and HPMC in the gel decreased the rate of release of ceftiofur to some extent. A decrease in the temperatures of the release medium decreased the release rate of ceftiofur, but not the rate of gel dissolution. The pH of the release medium showed a very slight effect on the release of ceftiofur and did not affect gel dissolution due to the non-ionic nature of P407.

Multi-unit controlled release systems of nifedipine and nifedipine:pluronic F-68 solid dispersions: characterization of release mechanisms.

Nifedipine (N) and nifedipine. Pluronic F-68 solid dispersion (SD) pellets were developed and characterizedfor drug release mechanisms from a multi-unit erosion matrix system for controlled release. Nifedipine was micronized using a jet mill. Solid dispersion with Pluronic F-68 was prepared by the fusion method. Nifedipine and SD were characterized by particle size analysis, solubility, differential scanning calorimetry (DSC), and x-ray diffraction (XRD) studies. Samples were subsequently processed into matrix pellets by extrusion/spheronization using Eudragit L 100-55 and Eudragit S 100 as release rate-controlling polymers. Drug release mechanisms from pellets were characterized by microscopy and mercury intrusion porosimetry; DSC and XRD studies indicated no polymorphic changes in N after micronization and also confirmed the formation of SD of N with Pluronic F-68. Pellets of N showed a 24-hr drug release profile following zero-order kinetics. Pellets of SD showed a 12-hr release profile followingfirst-order kinetics. Aqueous solubility of N after SD formation was found to be increased 10-fold. Due to increased solubility of N in SD, the drug release mechanism from the multi-unit erosion matrix changed from pure surface erosion to an erosion/diffusion mechanism, thereby altering the release rate and kinetics.

Investigation of the utility of an in vitro release test for optimizing semisolid dosage forms.

Current literature indicates that an in vitro release test (IVRT) can serve as a research tool during the course of developing topical formulations. The purpose of this study was therefore to investigate the ability of an IVRT to select the topical semisolid formulations with the most rapid release rate of the model drug ketoprofen from two closely related hydrogels in a simulated product development process. Two glycols with distinct differences in their physical-chemical properties, Transcutol P (ethoxydiglycol) and propylene glycol, were incorporated into Carbopol 980 and Poloxamer 407 formulations. The release rate of ketoprofen was determined utilizing different receptor media and conditions, i.e., phosphate buffer pH 7.4, isopropyl myristate (IPM), and a combination of an IPM soaked membrane and phosphate buffer (pH 7.4) as receptor fluid. The results indicated that the conditions chosen could affect greatly the conclusions concerning the formulations. The only observable trend was that Transcutol P-containing formulations tended to permit a faster ketoprofen release than propylene glycol-containing formulations when utilizing IPM as a receptor component. This was attributed to the mutual miscibility of Transcutol P in IPM. It can be concluded that, for the purpose of formulation screening in the early phases of product development, an IVRT will only be useful for predicting the amount of drug available for absorption if the receptor medium has properties that closely mimic human skin. These results illustrate the importance of selecting suitable receptor components and indicate that it may be necessary to consider alternatives to the commonly used synthetic membranes.