Delivery of doxorubicin loaded P18 conjugated-poly(2-ethyl-oxazoline)-DOPE nanoliposomes for targeted therapy of breast cancer.

Breast cancer, a heterogeneous disease, has the highest incidence rate and is a major cause of death in females worldwide. Drug delivery by using nanotechnology has shown great promise for improving cancer treatment. Nanoliposomes are known to have enhanced accumulation ability in tumors due to prolonged systemic circulation. Peptide 18 (P18), a tumor homing peptide targeting keratin-1 (KRT-1), was previously shown to have high binding affinity towards breast cancer cells. In this study, we investigate the ability of P18 conjugated PEtOx-DOPE nanoliposomes (P18-PEtOx-DOPE) for the targeted delivery of doxorubicin to AU565 breast cancer model. Toxicology studies of PEtOx-DOPE nanoliposomes performed on normal breast epithelial cells (MCF10A), showed minimal toxicity. Doxorubicin delivery by P18-PEtOx-DOPE to AU565 cells induces cytotoxicity in a dose and time dependent manner causing mitotic arrest in G2/M phase at 24 h. Anti-cancer activity of P18-PEtOx-DOPE-DOX nanoliposomes on AU565 cells was detected by Annexin V/PI apoptosis assay. In terms of in vivo antitumor efficacy, P18-PEtOx-DOPE-DOX nanoliposomes administration to AU565 CD-1 nu/nu mice model showed significant decrease in tumor volume suggesting that DOX delivered by these nanoliposomes elicited a strong antitumor response comparable to the free delivery of doxorubicin. Overall, our results offered preclinical proof for the use of P18-PEtOx-DOPE-DOX nanoliposomes in KRT-1+ breast cancer therapy.

Development of Zoledronic Acid Containing Biomaterials for Enhanced Guided Bone Regeneration.

In recent years, biomaterial-based treatments, also called guided bone regeneration (GBR), which aim to establish a bone regeneration site and prevent the migration of gingival connective tissue and / or peripheral epithelium through the defective area during periodontal surgical procedures have come to the fore. In this report, we have developed a nanoparticle bearing thermosensitive in situ gel formulation of Pluronic F127 and poly(D,L-lactic acid) based membrane to reveal their utilization at GBR by in-vivo applications. In addition, the encouragement of the bone formation in defect area via inhibition of osteoclastic activity is intended by fabrication these biodegradable biomaterials at a lowered Zoledronic Acid (ZA) dose. Both of the developed materials remained stable under specified stability conditions (25 °C, 6 months) and provided the extended release profile of ZA. The in-vivo efficacy of nanoparticle bearing in situ gel formulation, membrane formulation and simultaneous application for guided bone regeneration was investigated in New Zealand female rabbits with a critical size defect of 0.5 × 0.5 cm in the tibia bone for eight weeks. Based on the histopathological findings, lamellar bone and primarily woven bone formations were observed after 8 weeks of post-implantation of both formulations, while fibrosis was detected only in the untreated group. Lamellar bone growth was remarkably achieved just four weeks after the simultaneous application of formulations. Consequently, the simultaneous application of ZA-membrane and ZA-nanoparticles loaded in-situ gel formulations offers enhanced and faster GBR therapy alternatives.

Design and evaluation of peptide-18-targeted nanoliposomes constructed by poly(2-oxazoline)-DOPE for doxorubicin delivery.

AIMS: The aim of this study is to develop targeted nanoliposome formulations to provide efficient treatment for breast cancer. In this study, peptide 18-modified poly(2-ethyl-2-oxazoline)-dioleoylphosphatidylethanolamine (P18-PEtOx-DOPE), was synthesised to construct nanoliposomes. METHODS: Doxorubicin (DOX) was encapsulated into the nanoliposomes by ethanol injection method. Particle size and polydispersity index were measured by dynamic light scattering. Zeta potential was determined by electrophoretic laser Doppler anemometry. The shape of the nanoliposomes was examined by transmission electron microscope. Specific bindings of P18-PEtOx-DOPE nanoliposomes were demonstrated on AU565 cells by confocal microscopy and flow cytometry studies. RESULTS: DOX-loaded nanoliposomes with particle diameter of 150.00 ± 2.84 nm and PDI of 0.212 ± 0.013 were obtained. PEtOx-DOPE and PEtOx-DOPE nanoliposomes are non-toxic on HUVEC, HEK293 and hMSC cells for 48 h. Furthermore, P18-PEtOx-DOPE nanoliposomes demonstrated specificity towards AU565 cells with high binding affinity. CONCLUSIONS: As a result, DOX-loaded P18-PEtOx-DOPE nanoliposomes can serve as favourable candidates in breast cancer targeted therapy.

Guided bone regeneration by the development of alendronate sodium loaded in-situ gel and membrane formulations.

Biocompatible materials applied in guided bone regeneration are needed to prevent leakage caused by the invasion of peripheral epithelium. (2.1) The aim of this study is to develop a thermosensitive in situ gel system containing alendronate sodium loaded PLGA nanoparticles and alendronate sodium loaded membranes for guided bone regeneration. Thermosensitive Pluronic F127 gel system was preferred to prevent soft tissue migration to the defect site and prolong the residence time of the nanoparticles in this region. In situ gel system was combined with membrane formulation to enhance bone regenaration activity. Efficacy of combination system was investigated by implanting in 0.5 × 0.5 cm critical size defect in tibia of New Zealand female rabbits. According to the histopathological results, fibroblast formations were found at defect area after 6 weeks of post implantation. In contrast, treatment with the combination of in-situ gel containing nanoparticles with membrane provided woven bone formation with mature bone after 4 weeks of post implantation. As a results, the combination of in-situ gel formulation containing alendronate sodium-loaded nanoparticles with membrane formulation could be effectively applided for guided bone regeneration.

Design of ciprofloxacin-loaded nano-and microcomposite particles for dry powder inhaler formulations: preparation, in vitro characterisation, and antimicrobial efficacy.

In this study, ciprofloxacin hydrochloride (CIP)-loaded poly-ε-caprolactone (PCL) nanoparticles were prepared for pulmonary administration. CIP-loaded PCL nanoparticles were prepared using solid-in-oil-in-water (s/o/w) emulsion solvent evaporation method, and the effects of various formulation parameters on the physicochemical properties of the nanoparticles were investigated. PCL nanoparticles showed spherical shapes with particle sizes around 143-489 nm. Encapsulation efficiency was found to be very low because of water-solubility properties of CIP. However, the surface modification of nanoparticles with chitosan caused an increase in the encapsulation efficiency of nanoparticles. At drug release study, CIP-loaded PCL nanoparticles showed initial burst effect for 4 h and then continuously released for 72 h. Nanocomposite microparticles containing CIP-loaded PCL nanoparticles were prepared freeze-drying method and mannitol was used as carrier material. Tapped density and MMADt results show that nanocomposite microparticles have suitable aerodynamic properties for pulmonary administration. Antimicrobial efficacy investigations showed that CIP-encapsulated PCL nanoparticles and nanocomposite microparticles inhibited the growth of bacteria. Also, when the antimicrobial activity of the nanoparticles at the beginning and at the sixth month was examined, it was found that the structure of the particulate system was still preserved. These results indicated that nanocomposite microparticles containing CIP-loaded PCL nanoparticles can be used for pulmonary delivery.

Co-delivery of pemetrexed and miR-21 antisense oligonucleotide by lipid-polymer hybrid nanoparticles and effects on glioblastoma cells.

Combination therapy using anticancer drugs and nucleic acid is a more promising strategy to overcome multidrug resistance in cancer and to enhance apoptosis. In this study, lipid-polymer hybrid nanoparticles (LPNs), which contain both pemetrexed and miR-21 antisense oligonucleotide (anti-miR-21), have been developed for treatment of glioblastoma, the most aggressive type of brain tumor. Prepared LPNs have been well characterized by particle size distribution and zeta potential measurements, determination of encapsulation efficiency, and in vitro release experiments. Morphology of LPNs was determined by transmission electron microscopy. LPNs had a hydrodynamic size below 100 nm and exhibited sustained release of pemetrexed up to 10 h. Encapsulation of pemetrexed in LPNs increased cellular uptake from 6% to 78%. Results of confocal microscopy analysis have shown that co-delivery of anti-miR-21 significantly improved accumulation of LPNs in the nucleus of U87MG cells. Nevertheless, more effective cytotoxicity results could not be obtained due to low concentration of anti-miR-21, loaded in LPNs. We expect that the effective drug delivery systems can be obtained with higher concentration of anti-miR-21 for the treatment of glioblastoma.

Lysozyme-loaded lipid-polymer hybrid nanoparticles: preparation, characterization and colloidal stability evaluation.

CONTEXT: Lipid-polymer hybrid nanoparticles (LPNPs) are polymeric nanoparticles enveloped by lipid layers, which have emerged as a potent therapeutic nanocarrier alternative to liposomes and polymeric nanoparticles. OBJECTIVE: The aim of this work was to develop, characterize and evaluate LPNPs to deliver a model protein, lysozyme. MATERIALS AND METHODS: Lysozyme-loaded LPNPs were prepared by using the modified w/o/w double-emulsion-solvent-evaporation method. Poly-ɛ-caprolactone (PCL) was used as polymeric core material and tripalmitin:lechitin mixture was used to form a lipid shell around the LPNPs. LPNPs were evaluated for particle size distribution, zeta potential, morphology, encapsulation efficiency, in vitro drug release, stability and cytotoxicity. RESULTS: The DLS measurement results showed that the particle size of LPNPs ranged from 58.04 ± 1.95 nm to 2009.00 ± 0.52 nm. The AFM and TEM images of LPNPs demonstrate that LPNPs are spherical in shape. The protein-loading capacity of LPNPs ranged from 5.81% to 60.32%, depending on the formulation parameters. LPNPs displayed a biphasic drug release pattern with a burst release within 1 h, followed by sustained release afterward. Colloidal stability results of LPNPs in different media showed that particle size and zeta potential values of particles did not change significantly in all media except of FBS 100% for 120 h. Finally, the results of a cellular uptake study showed that LPNPs were significantly taken up by 83.3% in L929 cells. CONCLUSION: We concluded that the LPNPs prepared with PCL as polymeric core material and tripalmitin:lechitin mixture as lipid shell should be a promising choice for protein delivery.

Development of reconstitutable suspensions containing diclofenac sodium-loaded microspheres for pediatric delivery.

Effective clinical utilisation of non-steroidal anti-inflammatory drugs, such as diclofenac sodium (DS) is significantly limited by their ulcerogenic potential and poor bioavailability after oral administration. The objective of this work was to develop reconstitutable pediatric suspensions of DS-loaded microspheres prepared with an acrylic polymer (Eudragit RS) for improved pediatric delivery of DS. The microspheres were prepared by the water-in-oil-in-water or solid-in-oil-in-water emulsion techniques. Enviromental scanning electron microscopy observations clearly showed that microspheres have spherical shape. The drug entrapment efficiency of these microspheres was found 47.96 ± 0.79% to 88.57 ± 0.59% and their average particle sizes were 23.94-60.78 µm, which are within the desired range for the development of suspension formulation. The in vitro dissolution indicated prolonged sustained release of DS over 8 h. The results of preliminary characterisation studies of suspensions show that a liquid pharmaceutical preparation for oral administration capable of providing a sustained release of DS was successfully obtained.

Fast determination of diphenhydramine hydrochloride in reconstitutable syrups by CWT, PLS AND PCR methods.

Diphenhydramine hydrochloride (DPH), a histamine H1-receptor antagonist, is widely used as antiallergic, antiemetic and antitussive drug found in many pharmaceutical preparations. In this study, a new reconstitutable syrup formulation of DPH was prepared because it is more stable in solid form than that in liquid form. The quantitative estimation of the DPH content of a reconstitutable syrup formulation in the presence of pharmaceutical excipients, D-sorbitol, sodium citrate, sodium benzoate and sodium EDTA is not possible by the direct absorbance measurement. Therefore, a signal processing approach based on continuous wavelet transform was used to determine the DPH in the reconstitutable syrup formulations and to eliminate the effect of excipients on the analysis. The absorption spectra of DPH in the range of 5.0-40.0 µg/mL were recorded between 200-300 nm. Various wavelet families were tested and Biorthogonal1.1 continuous wavelet transform (BIOR1.1-CWT) was found to be optimal signal processing family to get fast and desirable determination results and to overcome excipient interference effects. For a comparison of the experimental results obtained by partial least squares (PLS) and principal component regression (PCR) methods were applied to the quantitative prediction of DPH in the mentioned samples. The validity of the proposed BIOR1.1-CWT, PLS and PCR methods were achieved analyzing the prepared samples containing the mentioned excipients and using standard addition technique. It was observed that the proposed graphical and numerical approaches are suitable for the quantitative analysis of DPH in samples including excipients.

Preparation and in vitro evaluation of surface-modified poly (lactide-co-glycolide) microparticles as biodegradable drug carriers for pulmonary peptide and protein delivery.

The present study reports the preparation and physicochemical characterization of surface-modified poly(lactide-co-glycolide) (PLGA) microparticles containing interleukin-2 (rhIL-2) for pulmonary delivery. The surface of the microparticles was modified with mucoadhesive polymers such as chitosan and Carbopol 971P. The feasibility of this surface modification was confirmed by measuring the zeta potential. Chitosan-modified PLGA microparticles showed a positive zeta potential, while Carbopol-modified PLGA microparticles were negatively charged. The mucin binding efficiency values have shown that the positively charged chitosan coated microparticles showed a higher adhesive percent to the mucin than the negatively charged un-coated or Carbopol 971P coated microparticles. Furthermore, surface modification of microparticles with chitosan and Carbopol 971P has yielded a slight decrease in the amount of protein initially released. These findings suggest the suitability of surface-modified PLGA microparticles as an efficient carrier system for delivery peptides and proteins to the respiratory tract.

Preparation and evaluation of double-walled microparticles prepared with a modified water-in-oil-in-oil-in-water (w1/o/o/w3) method.

In this study, a modified water-in-oil-in-oil-in-water (w1/o/o/w3) method was developed to prepare double-walled microparticles containing ovalbumin (OVA). The microparticles were characterized with respect to their morphology, particle size, encapsulation efficiency, production yield, thermal properties and in vitro drug release. Microscopy observations clearly showed that microparticles have spherical shape and smooth surface. These microparticles were characterized to have double-walled structure, with a cavity in the centre. By using w1/o/o/w3 method, a significant decrease in mean particle size and a significant increase in encapsulation efficiency were obtained. The mean particle size and the encapsulation efficiency of double-walled microparticles were also affected by the changing amount of OVA and mass ratio of polymers. Microparticles prepared with two polymers exhibited a significantly lower initial burst release followed by sustained release compared to microparticles made from poly(d,l-lactide-co-glycolide) 50/50 only. It can be concluded that these microparticles can be a potential delivery system for therapeutic proteins.

Preparation and evaluation of PLGA microparticles as carrier for the pulmonary delivery of rhIL-2 : I. Effects of some formulation parameters on microparticle characteristics.

In this study, recombinant human interleukin-2 (rhIL-2) containing poly(lactic-co-glycolic acid) (PLGA) microparticles were prepared for pulmonary administration by modified w/o/w double emulsion solvent extraction method and the effects of various formulation parameters on the physicochemical properties of the microparticles were investigated. Microparticles in suitable size for pulmonary administration (4.02 µm) were obtained by increasing dichloromethane volume used in the organic phase. Also, a very high encapsulation efficiency (99.22%) value could be reached in these microparticles. In the sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis, rhIL-2 extracted from microparticles having a similar band with native rhIL-2 showed that the protein was not affected by the encapsulation process. The release curves of microparticles exhibited a biphasic fashion, characterized by a fast release phase at initial 1 day, followed by a slower one on the remaining days. Bioactivity investigations using T cells show that rhIL-2 encapsulated in PLGA microparticles retain their biological activity.

Formulation and evaluation of reconstitutable suspensions containing ibuprofen-loaded Eudragit microspheres.

The objective of this work was to develop and evaluate reconstitutable suspensions of ibuprofen-loaded microspheres prepared with an acrylic polymer (Eudragit RS-PM). The microspheres were prepared by the quasi-emulsion solvent diffusion technique. To prepare reconstitutable suspension formulation, the microspheres used had a mean particle size of 316.6 microm and 99.8% loading efficiency. Xanthan gum was chosen as the suspending agent for the suspension formulations. D-sorbitol was used to impart palatability of suspensions. The amount of D-sorbitol affected sedimentation volume and redispersibility properties of suspensions. The highest improving effect was shown with 20.0% and 25.0% of D-sorbitol concentrations. It was observed that dispersion media of suspensions showed non-Newtonian flow characteristics. To ensure minimum drug leakage from the microspheres into the suspension, the pH was buffered at 3.60 using citrate buffer. The ibuprofen content calculated from the suspended microspheres was consistent with that from microspheres alone. This result indicated that no leakage of drug occurred from the microspheres in the suspension on storage. Moreover, the same release rate of ibuprofen from the microspheres suspension and microspheres alone indicated that the suspension medium studied did not affect the property of drug release. This study suggested that stable suspensions of ibuprofen-loaded microspheres could be formulated with 0.6% w/v xanthan gum by the addition of 20% w/v D-sorbitol.

Preparation and evaluation of poly(lactic-co-glycolic acid) microparticles as a carrier for pulmonary delivery of recombinant human interleukin-2: II. In vitro studies on aerodynamic properties of dry powder inhaler formulations.

OBJECTIVE: The aim of this study was the preparation and evaluation of dry powder formulations of recombinant human interleukin-2 (rhIL-2)-loaded microparticles to be administered to the lung by inhalation. METHODS: As indicated in our previous study, the microparticles were prepared by modified water-in-oil-in-water (w(1)/o/w(3)) double emulsion solvent extraction method using poly(lactic-co-glycolic acid) (PLGA) polymers. The dry powder formulations were prepared with blending of microparticles and mannitol as a coarse carrier. The actual aerodynamic characteristics of the microparticles alone and prepared mixtures with mannitol are evaluated by using the eight-stage Andersen cascade impactor. RESULTS: Due to the low tapped density of microparticles (<0.4 g/cm(3)), the theoretical aerodynamic diameter (MMADt) values were calculated (<5 µm) on the basis of the geometrical particle diameter and tapped density values. The lowest tapped density value (0.17 g/cm(3)) belongs to the cyclodextrin-containing formulation. According to the results obtained using the cascade impactor, the emitted doses for all microparticle formulations were found to be rather high and during the aerosolization for all the formulations except F3 and F5, >90% of the capsule content was determined to be released. However, the actual aerodynamic diameter (MMADa) values were seen to be higher than the MMADt values. The blending of the microparticles with mannitol allowed their aerodynamic diameters to decrease and their fine particle fraction values to increase. CONCLUSION: The obtained results have shown that the mixing of rhIL-2-loaded microparticles with mannitol possess suitable aerodynamic characteristics to be administered to the lungs by inhalation.

Preparation and evaluation of modified release ibuprofen microspheres with acrylic polymers (Eudragit) by quasi-emulsion solvent diffusion method: effect of variables.

The aim of this study was to prepare and evaluate microspheres containing ibuprofen. Microspheres were prepared by modified quasi-emulsion solvent diffusion method. The influence of formulation factors (drug-polymer ratio, volumes of solvent, polyvinyl alcohol concentration and type of polymer) on the morphology, particle size distribution, drug loading capacity, micromeritical properties and the in vitro release characteristics of the microspheres were investigated. Physical characterizations of ibuprofen microspheres were also carried out using scanning electron microscopy, X-ray diffractometry and IR spectrophotometry. It was found that the yield of preparation was dependent on the initial temperature gradient between the emulsion phases. When there was an initial difference of temperature between the aqueous phase and dispersed emulsion phases, yield of preparation was increased distinctly. The drug loading capacities were very high for all formulations of the microspheres which were obtained. Mean particle size changed by changing the drug-polymer ratio, volumes of solvent or polyvinyl alcohol concentration. The flow properties were much improved over those of the original crystals. In vitro dissolution results showed that the release rate of ibuprofen was modified in all formulations. Although ibuprofen release rates from Eudragit RS microspheres were very slow, they were fast from Eudragit RL microspheres. These results observed that if Eudragit RS and Eudragit RL are used in combination, optimum release profiles may be obtained.