Unlocking apoptosis in triple negative breast cancer: Harnessing "glutamine trap" to amplify the efficacy of lapatinib-loaded mixed micelles.

Certain aggressive cancers, such as triple-negative breast cancer (TNBC), heavily bank on glutamine for their proliferation and survival. In this context, TNBC functions as a "glutamine trap," extracting circulating glutamine at a rate surpassing that of any other organ. Moreover, the overexpression of Alanine, Serine, Cysteine Transporter 2 (ASCT2), a key player in glutamine uptake, further underscores the significance of targeted therapy to enhance TNBC treatment. This led to the exploration of a novel approach involving hydrophobized Pluronic-based mixed micelles achieved through the use of docosahexaenoic acid and stapled with glutamine for displaying inherent ASCT2 targeting ability-a formulation termed LPT G-MM. LPT G-MM exhibited optimal characteristics, including a size of 163.66 ± 10.34 nm, a polydispersity index of 0.237 ± 0.083, and an enhanced drug loading capacity of approximately 15 %. Transmission electron microscopy validated the spherical shape of these micelles. In vitro release studies demonstrated drug release in a sustained manner without the risk of hemolysis. Importantly, LPT G-MM displayed heightened cellular uptake, increased cytotoxicity, a lower IC50 value, elevated reactive oxygen species, induced mitochondrial membrane depolarization, and a greater apoptosis index in TNBC cell lines compared to free LPT. The pharmacokinetic profile of LPT G-MM revealed a substantial rise in half-life (t1/2) by approximately 1.48-fold and an elevation in the area under the curve [AUC(0→∞)] by approximately 1.19-fold. Moreover, there was a significant reduction in the percentage of tumor volume by approximately 7.26-fold, along with decreased serum toxicity markers compared to free LPT. In summary, LPT G-MM demonstrated promising potential in boosting payload capacities and targeting specificity in the context of TNBC treatment.

Exploring the synergistic behavior of paclitaxel and vorinostat upon co-loading in albumin nanoparticles for breast cancer management.

Breast cancer is challenging to treat accompanied with poor clinical outcomes. Paclitaxel (PTX) is a first-line chemotherapeutic agent, but possesses limitations due to side effects, high dose, non-specific tissue distribution, and drug resistance. An epigenetic modulator, vorinostat (VOR) is known to enhance PTX efficacy and therefore to resolve the issues of conventional PTX formulations, we designed PTX- and VOR-bound albumin nanoparticles (PTX-VOR-BSA-NPs) using antisolvent precipitation technique where albumin is used as a carrier and a targeting agent. The PTX-VOR-BSA-NPs were of 140 nm size, polydispersity index around 0.18, and about 78% and 68% of entrapment efficiency for PTX and VOR, respectively. A bi-pattern release of both PTX and VOR was observed from PTX-VOR-BSA-NPs with a burst release for 2 h succeeded by sustained release until 24 h. A significantly lower %cell viability was observed in MCF-7 cell lines, while efficient cellular drug uptake was found in MDA-MB-231 cells. Furthermore, a greater apoptotic index was found compared to free PTX and VOR because of the synergistic activity of these drugs. The PTX-VOR-BSA-NPs also showcased superior pharmacokinetic profile and noteworthy reduction in the tumor volume compared to Intaxel in 4T1 cell line-induced breast tumor model. Further, the NPs showed similar levels of toxicity biomarkers as that of control. Overall, the developed PTX-VOR-BSA-NPs were found to have less toxicity and more effectiveness compared to the marketed formulation, thus affirming the generation of a potent as well as and safe product.

Enhancing anti-cancer potential by delivering synergistic drug combinations via phenylboronic acid modified PLGA nanoparticles through ferroptosis-based therapy.

In this study, we investigated the potential of the sorafenib (SOR) and simvastatin (SIM) combination to induce ferroptosis-mediated cancer therapy. To enhance targeted drug delivery, we encapsulated the SOR + SIM combination within 4-carboxy phenylboronic acid (CPBA) modified PLGA nanoparticles (CPBA-PLGA(SOR + SIM)-NPs). The developed CPBA-PLGA(SOR + SIM)-NPs exhibited a spherical shape with a size of 213.1 ± 10.9 nm, a PDI of 0.22 ± 0.03, and a Z-potential of -22.9 ± 3.2 mV. Notably, these nanoparticles displayed faster drug release at acidic pH compared to physiological pH. In cellular experiments, CPBA-PLGA(SOR + SIM)-NPs demonstrated remarkable improvements, leading to a 2.51, 2.69, and 2.61-fold decrease in IC50 compared to SOR alone, and a 7.50, 16.71, and 5.11-fold decrease in IC50 compared to SIM alone in MDA-MB-231, A549, and HeLa cells, respectively. Furthermore, CPBA-PLGA(SOR + SIM)-NPs triggered a reduction in glutathione (GSH) levels, an increase in malondialdehyde (MDA) levels, and mitochondrial membrane depolarization in all three cell lines. Pharmacokinetic evaluation revealed a 2.50- and 2.63-fold increase in AUC0-∞, as well as a 1.53- and 2.46-fold increase in mean residence time (MRT) for SOR and SIM, respectively, compared to the free drug groups. Notably, the CPBA-PLGA(SOR + SIM)-NPs group exhibited significant reduction in tumor volume, approximately 9.17, 2.45, and 1.63-fold lower than the control, SOR + SIM, and PLGA(SOR + SIM)-NPs groups, respectively. Histological examination and biomarker analysis showed no significant differences compared to the control group, suggesting the biocompatibility of the developed particles for in-vivo applications. Altogether, our findings demonstrate that CPBA-PLGA(SOR + SIM)-NPs hold tremendous potential as an efficient drug delivery system for inducing ferroptosis, providing a promising therapeutic option for cancer treatment.

Synergistic anticancer therapy via ferroptosis using modified bovine serum albumin nanoparticles loaded with sorafenib and simvastatin.

Ferroptosis is a non-apoptotic cell death pathway characterized by the accumulation of lipid-peroxy radicals within the affected cells. Here, we investigate the synergistic capacity of sorafenib (SOR) and simvastatin (SIM) to trigger ferroptosis for cancer therapy. For precise in-vivo delivery, SOR + SIM was ratiometrically loaded in bovine serum albumin nanoparticles (BSA-NPs) modified with 4-carboxy phenylboronic acid (CPBA). The developed CPBA-BSA(SOR + SIM)-NPs revealed size of 175.2 ± 12.8 nm, with PDI of 0.22 ± 0.03 and Z-potential of -29.6 ± 4.8 mV. Significantly, CPBA-BSA(SOR + SIM)-NPs exhibited > 2 and > 5-fold reduction in IC50 values compared to individual SOR and SIM treatments respectively, in all tested cell lines. Moreover, CPBA-BSA(SOR + SIM)-NPs treated cells exhibited decrease in glutathione levels, increase in malonaldehyde levels and depolarization of mitochondrial membrane potential (JC-1 assay). Pharmacokinetic analysis revealed enhanced AUC0-∞ and MRT levels for SOR and SIM when administered as CPBA-BSA(SOR + SIM)-NPs compared to free drugs. Crucially, in in-vivo experiments, CPBA-BSA(SOR + SIM)-NPs led to a significant reduction in tumor volume compared to various control groups. Histological and biomarker analyses underscore their biocompatibility for clinical applications. In conclusion, this study highlights the potential of CPBA-BSA(SOR + SIM)-NPs as a promising strategy for inducing ferroptosis in cancer cells, concurrently improving drug delivery and therapeutic efficacy. This approach opens new avenues in cancer treatment.

Enhancing oral bioavailability of insulin through bilosomes: Implication of charge and chain length on apical sodium-dependent bile acid transporter (ASBT) uptake.

This study investigates the impact of charge and chain length of bile salts in the bilosomes on the oral bioavailability of insulin (IN) by examining their uptake via the apical sodium-dependent bile acid transporter (ASBT). Deoxycholic acid bile salt was conjugated with different amino acids to create conjugates with varying charge and chain length, which were then embedded in liposomes. The resulting bilosomes had a particle size <400 nm, a PDI of 0.121 ± 0.03, and an entrapment efficiency of ∼70 %, while maintaining the chemical and conformational integrity of the loaded IN. Bilosomes also provided superior protection in biological fluids without compromising their biophysical attributes. Quantitative studies using the Caco-2 cell line demonstrated that anionic bilosomes were taken up more efficiently through ASBT than cationic bilosomes with 4- and 1.3-fold increase, respectively. Ex-vivo permeability studies corroborated these findings. In-vivo efficacy studies revealed a 1.6-fold increase in the AUC of IN with bilosomes compared to subcutaneous IN. The developed bilosomes were able to reduce blood glucose levels by ∼65 % at 6 h, with a cumulative hypoglycemic value of 35 % and a BAR of ∼30 %. These results suggest that ASBT can be a suitable target for improving the oral bioavailability of bilosomes containing IN.

Engineered Nano-carrier systems for the oral targeted delivery of follicle stimulating Hormone: Development, characterization, and, assessment of in vitro and in vivo performance and targetability.

Follicle stimulating hormone (FSH) is widely used for the treatment of female infertility, where the level of FSH is suboptimal due to which arrest in follicular development and anovulation takes place. Currently, only parenteral formulations are available for FSH in the market. Due to the drawbacks of parenteral administration and the high market shares of FSH, there is a need for easily accessible oral formulation. Therefore, enteric coated capsules filled with FSH loaded nanostructured lipid carriers (NLCs) or liposomes were prepared. Preliminary studies such as circular dichroism, SDS-PAGE, FTIR and ELISA were conducted to analyze FSH. Prepared formulations were optimized with respect to the size, polydispersity index, zeta potential, and entrapment efficiency using the design of experiments. Optimized formulations were subjected to particle counts and distribution analysis, TEM analysis, in vitro drug release, dissolution of enteric coated capsules, cell line studies, everted sac rat's intestinal uptake study, pharmacokinetics, pharmacodynamics, and stability studies. In the case of liposomes, RGD conjugation was done by carbodiimide chemistry and conjugation was confirmed by FTIR, 1HNMR and Raman spectroscopy. The prepared formulations were discrete and spherical. The release of FSH from enteric coated capsules was slow and sustained. The increased permeability of nano-formulations was observed in Caco-2 monoculture as well as in Caco-2 and Raji-B co-culture models. NLCs and liposomes showed an improvement in oral bioavailability and efficacy of FSH in rats. This may be due to mainly chylomicron-assisted lymphatic uptake of NLCs; whereas, in the case of liposomes, RGD-based targeting of ß1 integrins of M cells on Peyer's patches may be the main reason for the better effect by FSH. FSH was found to be stable chemically and conformationally. Overall, the study reveals the successful development and evaluation of FSH loaded NLCs and liposomes.

Exploring paclitaxel-loaded adenosine-conjugated PEGylated PLGA nanoparticles for targeting triple-negative breast cancer.

In present investigation, we developed paclitaxel (PTX)-loaded adenosine (ADN)-conjugated PLGA nanoparticles for combating triple-negative breast cancer (TNBC), where ADN acts as a substrate for adenosine receptors (AR) that are overexpressed in TNBC. Using synthesized PLGA-PEG-ADN, PTX-loaded nanoparticles (PTX ADN-PEG-PLGA NPs) were prepared via emulsion diffusion evaporation process that rendered particles of size 135 ± 12 nm, PDI of 0.119 ± 0.03, and entrapment-efficiency of 79.26 ± 2.52%. The NPs showed higher %cumulative release at pH 5.5 over 7.4 with Higuchi release kinetics. The PTX ADN-PEG-PLGA NPs showed ~ 4.87- and 5.22-fold decrease in %hemolysis in comparison to free PTX and Intaxel®, indicating their hemocompatible nature. The ADN modification assisted cytoplasmic internalization of particles via AR-mediated endocytosis that resulted in ~ 3.77- and 3.51-fold reduction in IC50 and showed apoptosis index of 0.93 and 1.18 in MDA-MB-231 and 4T1 cells respectively. The pharmacokinetic profile of ADN-PEG-PLGA NPs revealed higher AUC and t1/2 than Intaxel® and Nanoxel® pharmacodynamic activity showed ~ 18.90-fold lower %tumor burden than control. The kidney and liver function biomarkers showed insignificant change in the levels, when treated with PTX ADN-PEG-PLGA NPs and exhibited no histological alterations in the liver, spleen, and kidney. Overall, the optimized particles were found to be biocompatible with improved anti-TNBC activity.

Self-nanoemulsifying formulation for oral delivery of sildenafil: effect on physicochemical attributes and in vivo pharmacokinetics.

Sildenafil (SLD) is employed for the management of erectile dysfunction and pulmonary arterial hypertension. It exhibits meagre water solubility and is available in the form of citrate salt hydrate to improve the solubility. However, it still exhibits moderate solubility, high first-pass metabolism, resulting in very less oral bioavailability. The present study demonstrates the preparation of self-nanoemulsifying drug delivery system for augmenting the oral bioavailability of SLD. Oleic acid and Capmul MCM C8 blend (oil phase), Cremophor® RH40 (surfactant), and Labrafil® M1944 CS (cosurfactant) were selected as main constituents for making liquid preconcentrate based on the solubility and emulsification study. The preconcentrate upon dilution and emulsification showed droplet size 52.03 ± 13.03 nm, PDI 0.143 ± 0.028, and % transmittance was 99.77 ± 1.86% with SLD load of 40 mg/g of formulation. The prepared formulation was further assessed for stability, in vitro release, Caco-2 cell uptake, and in vivo pharmacokinetic performance. SLD-SNEDDS formulation was found to be robust in terms of stability against several folds dilution in the gastrointestinal tract (GIT), freeze-thaw cycles, and had a storage stability of 3 months at 4 °C and 25 °C. SLD-SNEDDS showed ~4.7-fold and ~5-fold increase in time- and concentration-dependent cellular uptake as against SLD cultured with Caco-2 cells. In vivo pharmacokinetic study revealed ~5.8- and ~2.5-fold increase in AUC0-∞ values in case of SLD-SNEDDS as against SLD suspension and SLD citrate solution, respectively.

Unfolding the Potency of Adenosine in Targeting Triple Negative Breast Cancer via Paclitaxel-Incorporated pH-Responsive Stealth Liposomes.

Triple-negative breast cancer (TNBC) belongs to the category of the most destructive forms of breast cancer. Being a highly potent chemotherapeutic agent, paclitaxel (PTX) is extensively utilized in the management of various cancers. Commercially available PTX formulations contain non-targeted drug carriers that result in low antitumor activity because of non-specific tissue distribution. Thus, to resolve this issue, we designed PTX-loaded pH-sensitive liposomes (pH Lipos) in the present investigation and used adenosine (ADN) as a targeting ligand. Further, d-α-tocopheryl polyethylene glycol succinate (TPGS) was incorporated into the liposomes to impart a stealth effect to the system. For the development of these pH Lipos, different conjugates were synthesized (ADN-CHEMS and TPGS-ADN) and further utilized for the preparation of ADN-PEG-pH Lipo and ADN-pH Lipo by a thin-film hydration method. DOPE:HSPC:CHEMS:cholesterol at a molar ratio of 3:3:2:2 was selected for the preparation of pH-Lipo possessing 7.5% w/w drug loading. They showed a particle size below 140 nm, a PDI below 0.205, and a % EE greater than 60%. All of the pH Lipos displayed a biphasic pattern of PTX release at pH 7.4 and 5.5. However, the percent drug release at pH 5.5 was substantially greater because of the pH-sensitive nature of the liposomes. The MDA MB 231 and 4T1 cell lines depicted improvement in the qualitative as well as quantitative cellular uptake of PTX ADN-PEG-pH Lipo with a substantial decrease in the IC50 value. Moreover, a higher apoptotic index was observed with pH Lipo compared to free PTX. PTX ADN-PEG-pH Lipo revealed a 3.98- and 3.41-fold rise in the AUC and t1/2 values of PTX compared to Intaxel, respectively. Overall, characteristic decreases in tumor volume and serum toxicity marker levels were observed, which confirmed the development of an efficient and safe formulation.

Hitting Multiple Cellular Targets in Triple-Negative Breast Cancer Using Dual-Action Cisplatin(IV) Prodrugs for Safer Synergistic Chemotherapy.

Triple-negative breast cancer (TNBC) cells show improved sensitivity for cisplatin therapy due to their defective DNA damage repair system. However, the clinical utilization of cisplatin is limited by dose-dependent systemic toxicities and chemoresistance. Cisplatin Pt(IV) derivatives having kinetically inert octahedral geometry provide an effective strategy to overcome these limitations. Upon cellular reduction, these derivatives release cisplatin and axial ligands, acting as dual-action prodrugs. Hereby, we have developed three cisplatin(IV) conjugates using distinct bioactive axial moieties (valproate, tocopherol, and chlorambucil), which can synergistically complement cisplatin activity and attack multiple cellular targets. The designed derivatives showcased enhanced antiproliferative activity and improved therapeutic synergism along with a noteworthy cisplatin dose reduction index in a panel of six cancer cells. These Pt(IV) derivatives remarkably improved cellular drug uptake and showed lower dependency on copper transporter 1 (Ctr1) for uptake than cisplatin. The results of enhanced in vitro activity were well corroborated by in vivo efficacy testing in the 4T1 cell-based TNBC model, showcasing ∼2-7-folds higher tumor volume reduction for Pt(IV) derivatives than cisplatin. In addition, the designed derivatives significantly reduced the nephrotoxicity risk involved in cisplatin therapy, indicated by systemic toxicity biomarkers and organ histopathology. The results indicated that cisplatin(IV) derivatives could open new avenues for safer synergistic chemotherapy in TNBC.

Partial inclusion complex assisted crosslinked ß-cyclodextrin nanoparticles for improving therapeutic potential of docetaxel against breast cancer.

The present investigation demonstrates the development of crosslinked ß-cyclodextrin nanoparticles (ß-CD NPs) for enhancing the therapeutic efficacy of docetaxel (DTX) against breast cancer. Initially, a partial inclusion complex between ß-CD and polypropylene glycol (PPG) was formed to induce self-assembly. This was followed by crosslinking of ß-CDs using epichlorohydrin (EPI) and removal (by solubilization) of PPG to yield uniform ß-CD NPs. The formed particles were used for loading DTX to form DTX ß-CD NPs. The resultant DTX ß-CD NPs exhibited particle size of 223.36 ± 17.73 nm with polydispersity index (PDI) of 0.13 ± 0.09 and showed entrapment efficiency of 54.53 ± 2%. Increased cell uptake (~5-fold), cytotoxicity (~3.3-fold), and apoptosis were observed in MDA-MB-231 cells when treated with DTX ß-CD NPs in comparison to free DTX. Moreover, pharmacokinetic evaluation of DTX ß-CD NPs revealed ~2 and ~5-fold increase in AUC0-∞ and mean residence time (MRT) of DTX when compared to Docepar®. Further, the anti-tumor activity using DMBA-induced cancer model showed that DTX ß-CD NPs were capable of reducing the tumor volume to ~40%, whereas Docepar® was able to reduce tumor volume till ~80%. Finally, the toxicity evaluation of DTX ß-CD NPs revealed no short-term nephrotoxicity and was confirmed by estimating the levels of biomarkers and histopathology of the organs. Thus, the proposed formulation strategy can yield uniformly formed ß-CD NPs which can be effectively utilized for improving the therapeutic efficacy of DTX.

Light-assisted anticancer photodynamic therapy using porphyrin-doped nanoencapsulates.

Light activatable porphyrinic photosensitizers (PSs) are essential components of anticancer and antimicrobial therapy and diagnostic imaging. However, their biological applications are quite challenging due to the lack of hydrophilicity and biocompatibility. To overcome such drawbacks, photosensitizers can be doped into a biocompatible polymer such as gelatin and further can be used for biomedical applications. Herein, first, a novel A4 type porphyrin PS [5,10,15,20-tetrakis(4-pyridylamidephenyl)porphyrin; TPyAPP] was synthesized via a rational route with good yield. Further, this porphyrin was encapsulated into the gelatin nanoparticles (GNPs) to develop hydrophilic phototherapeutic nanoagents (PTNAs, A4por-GNPs). Notably, the synthesis of such porphyrin-doped GNPs avoids the use of any toxic chemicals or solvents. The nanoprobes have also shown good fluorescence quantum yield demonstrating their applicability in bioimaging. Further, the mechanistic aspects of the anticancer and antimicrobial efficacy of the developed A4por-GNPs were evaluated via singlet oxygen generation studies. Overall, our results indicated porphyrin-doped biodegradable polymeric nanoparticles act as effective phototherapeutic agents against a broad range of cancer cell lines and microbes upon activation by the low-cost LED light.

Green surfactant-dendrimer aggreplexes: An ingenious way to launch dual attack on arch-enemy cancer.

Combination therapy, which combines anti-cancer drugs with different oligonucleotides, have shown potential in cancer treatment. However, delivering a hydrophobic anti-cancer drug and a hydrophilic oligonucleotide simultaneously is a herculean task. This study takes advantage of interactions between histidine-lauric acid-based green surfactant and poly(amidoamine) dendrimers to achieve this aim. The green surfactant was synthesized by carbodiimide chemistry and characterized by FTIR, 1H-NMR, and mass spectroscopy. Further, green surfactant-dendrimer aggregates encapsulating DTX and complexing SIRT 1 shRNA i.e., "aggreplexes" were developed and characterized. The term "aggreplexes" signifies complexes which are formed between green-surfactant-dendrimer aggregates and SIRT-1 shRNA via electrostatic interaction. The aggreplexes displayed particle size of 262.33 ± 3.87 nm, PDI of 0.25 and entrapment efficiency of 70.56 %. The TEM images revealed spherical shape of aggreplexes with irregular outer surface and corroborated particle size obtained from zetasizer. The in-vitro release study revealed biphasic release patterns of DTX from aggreplexes and were compatible for intravenous administration. Further, aggreplexes augmented cellular uptake in MDA-MB-231 cells by ∼1.87-fold compared to free DTX. Also, EGFP expression revealed significantly higher transfection of aggreplexes compared to naked shRNA and Superfect™ complexes. Further, aggreplexes showed higher cytotoxicity in MDA-MB-231 cells and ∼4.16-fold reduction in IC50 value compared to free DTX. Finally, apoptosis-index observed in case of aggreplexes was ∼3.57-fold higher than free DTX. These novel aggreplexes showed increased drug loading capacity and superior gene transfection potential. Thus, they open new avenues for co-delivery of hydrophobic anti-cancer drugs and hydrophilic therapeutic genes for improving current standards of cancer therapy.

pH sensitive liposomes assisted specific and improved breast cancer therapy using co-delivery of SIRT1 shRNA and Docetaxel.

Combining the bio-therapeutics with chemotherapeutic drugs can assist in augmenting the therapeutic standards by increasing the efficacy and decreasing the toxicity. Hence, in the present investigation Docetaxel (DTX) loaded pH-sensitive SIRT1 shRNA complexed lipoplex (DTX-lipoplex) were developed and explored for their improved breast cancer potential. The DTX-lipoplex were prepared by solvent evaporation and rehydration method and were evaluated for various quality attributes (particle size, % entrapment efficiency, hemotoxicity, DNA stability efficiency etc.), in vitro drug release, cell culture assays, antitumor efficacy and in vivo toxicity. The DTX-lipoplex exhibited a size of ~200 nm and zeta-potential of ~20 mV with ~70% encapsulation. Through systematic in vitro and in vivo examinations, DTX-lipoplex showed ~3 fold higher DTX titre within the tumor cells thereby significantly reducing the tumor burden (~78%) when compared to the marketed non pH sensitive lipid transfection agent and clinical counterpart i.e. Taxotere®. Thus, to conclude it can be said that co-delivering DTX and SIRT1 shRNA in a single tumor-specific nano-platform can improve the therapeutic potential of current therapy.

Tumor microenvironment responsive VEGF-antibody functionalized pH sensitive liposomes of docetaxel for augmented breast cancer therapy.

The present work exploits the tumor microenvironment which differs significantly from normal cellular environment in terms of both, having lower extracellular pH and increased angiogenesis capacity. To reduce systemic toxicity of docetaxel (DTX) and increase its therapeutic potential, VEGF antibody functionalized PEGylated pH sensitive liposomes (VEGF-PEG-pH-Lipo-DTX) were developed. The liposomes prepared by thin film hydration technique were later conjugated with VEGF antibody on liposomal surface by standard carbodiimide chemistry and using DSPE-PEG-COOH as linker. The VEGF-PEG-pH-Lipo-DTX displayed particle size of ~206 nm with an entrapment efficiency of ~62%. The transmission electron microscopy images revealed spherical shape of liposomes and corroborated the particle size obtained from zetasizer. The in vitro release study revealed biphasic release pattern of DTX from VEGF-PEG-pH-Lipo-DTX. The % drug released was also significantly higher at pH 5.5 which guarantees rapid endosomal escape and faster intracellular drug release. In case of VEGF-PEG-pH-Lipo-DTX the cellular uptake in MCF-7 cell line was augmented ~3.17-fold as compared to free DTX probably due to the VEGF-positive nature of MCF-7 cell (increased affinity for VEGF). Further, it was evident from the cytotoxicity assay that VEGF-PEG-pH-Lipo-DTX showed higher cytotoxicity in MCF-7 cells and ~5.78-fold reduction in IC50 value as compared to free DTX. The apoptotic index observed in case of VEGF-PEG-pH-Lipo-DTX was ~1.70-fold higher than free DTX. The VEGF-PEG-pH-Lipo-DTX inhibited the proliferation of HUVECs stimulated by VEGF, warranting its anti-angiogenic potential. Furthermore, pharmacokinetic profile of VEGF-PEG-pH-Lipo-DTX revealed a ~2.94-fold increase in t1/2 and a ~1.25-fold higher AUC (0→∞) as compared to marketed formulation Taxotere®. Similarly, mean residence time was also increased ~2.50-fold as compared to Taxotere®. Finally, treatment with VEGF-PEG-pH-Lipo-DTX demonstrated significant reduction in % tumor burden (~35%) as compared to Taxotere® (~75%). Thus, the combined approach of using PEGylated pH sensitive liposomes along with VEGF antibody functionalization for efficient targeting can improve current standards of DTX therapy for treatment of breast cancer.

Exploring protein stabilized multiple emulsion with permeation enhancer for oral delivery of insulin.

In present study, we have developed W/O/W microemulsion (ME) containing piperine (PiP) as a permeation enhancer and albumin (Alb) serving as a stabilizer for oral delivery of insulin (INS). The resultant formulation, ME(INS)-PiP-Alb exhibited droplet size of 3.35 ± 0.25 µm along with polydispersity index (PDI) of 0.30 ± 0.10. The formulation process employed for developing ME(INS)-PiP-Alb showed no effect on INS's chemical and conformational stability. Further, ME(INS)-PiP-Alb was able to maintain desired attributes (size & PDI) along with INS stability in simulated gastrointestinal fluids. Also, ME(INS)-PiP-Alb rendered higher protection to INS in presence of pepsin and trypsin than ME(INS)-PiP. In qualitative Caco-2 cell uptake, INS loaded ME's showed higher uptake in comparison to free INS. Whereas, in permeability studies ME(INS)-PiP-Alb showed ~4 and ~1.5-fold enhanced permeation than free INS and ME(INS) without PiP groups respectively. Also, in ex vivo intestinal permeation studies similar fold increment in permeation were observed. Interestingly, the pharmacodynamic studies revealed ~3.2-fold higher hypoglycemic effect in animals treated with ME(INS)-PiP-Alb in comparison to ME(INS)-PiP. Similarly, the pharmacokinetic studies also revealed ~1.6 fold higher AUC for ME(INS)-PiP-Alb than ME(INS)-PiP. Thus, in vivo results suggested that Alb as a stabilizer can assist in improving the hypoglycemic effect of the developed ME with PiP. Hence, this strategy can also be extrapolated for delivering other bio-macromolecules orally.

QbD-Steered Development of Biotin-Conjugated Nanostructured Lipid Carriers for Oral Delivery of Chrysin: Role of Surface Modification for Improving Biopharmaceutical Performance.

Bioactives are documented to exhibit diverse pharmacological activities, however, their low and inconsistent bioavailability primarily pose serious impediment against their potential therapeutic usage. Efforts, therefore, have been undertaken to systematically develop nanostructured lipidic carriers of chrysin, a vital flavonoid, employing Capmul PG-12 (i.e., liquid lipid), glyceryl monostearate (i.e., solid lipid), stearylamine, Phospholipid S-100 (i.e., cosurfactant) and Poloxamer 188 (i.e., surfactant). NLCs were formulated using hot-melt dispersion-high pressure homogenization method and optimized using Face-Centred Cubic Design. Afterwards, stearylamine was conjugated with biotin as ligand through EDC-NHS coupling reaction and biotin-staerylamine complex formation was ratified using H-1NMR and FTIR. It was further used instead of SA for the preparation of biotin-conjugated-optimized NLCs (Bio-NLCs). Mean particle size of consequent Bio-NLCs was found to be 246.4 nm and zeta potential as 11.4 mV. In vitro release studies indicated sustained drug release characteristics from NLCs over 48 h. Cell line studies conducted on coumarin6-loaded Bio-NLCs in demonstrated remarkably superior cellular uptake over naive NLCs and pure dye. Marked improvement in absorption parameters was observed during in vivo pharmacokinetics for Bio-NLCs and NLCs vis-à-vis pure chrysin suspension. However, the improvement for naive NLCs was relatively lower than that of Bio-NLCs. Almost 5.20-folds augmentation in Cmax (p < 0.005), 8.94-folds in AUC0-24 (p < 0.001), 7.46-folds in AUC0-∞ (p < 0.001) and 7.25-folds in Ka (p < 0.01), signify improved degree of drug absorption and retention of Bio-NLCs. Stability studies indicated the robustness of Bio-NLCs, when stored under refrigerated storage conditions for 3 months. By and large, the current work demonstrates high potential of Bio-NLCs for distinctly improved biopharmaceutical performance of chrysin.

Exploring the Promising Potential of High Permeation Vesicle-Mediated Localized Transdermal Delivery of Docetaxel in Breast Cancer To Overcome the Limitations of Systemic Chemotherapy.

The currently available systemic chemotherapy for treating breast cancer often results in serious systemic side effects and compromises patient compliance. The distinct anatomical features of human breasts (e.g., embryological origin of breast skin, highly developed internal lymphatic and venous circulation, and the presence of mammary fat layers) help in preferential accumulation of drugs into breasts after topical application on breast region. This unique feature is termed as localized transdermal delivery which could be utilized for effectively delivering anticancer agents to treat breast cancer and reducing the systemic side effects by limiting their presence in blood. However, the clinical effectiveness of this drug delivery approach is highly limited by barrier properties of skin reducing the permeation of anticancer drugs. In the present work, we have developed high permeation vesicles (HPVs) using phospholipids and synergistic combination of permeation enhancers (SCOPE) to improve the skin permeation of drugs. Docetaxel (DTX) was used as a model drug for hypothesis testing. The optimized SCOPE mixture composed of sodium oleate/sodium lauryl ether sulfate/propylene glycol in 64:16:20% w/w ratio. DTX HPVs were prepared using phospholipid: SCOPE, 8:2% w/w ratio. DTX HPVs exhibited as a uniform deformable vesicles with size range 124.2 ± 7.6 nm, significantly improved skin permeation profile, and sustained drug release until 48 h. Superior vesicle deformability, better vesicle membrane fluidization, and SCOPE mediated enhancement in skin fluidization were the prime factors behind enhancement of DTX permeation. The improved cellular uptake, reduced IC50 values, and higher apoptotic index of DTX HPVs in MCF-7 and MDA-MB-231 cells ensured the therapeutic effectiveness of HPV based therapy. Also, HPVs were found to be predominantly internalized inside cells through clathrin and caveolae-dependent endocytic pathways. Bioimaging analysis in mice confirmed the tumor penetration potential and effective accumulation of HPVs inside tumors after topical application. In vivo studies were carried out in comparison with marketed intravenous DTX injection (Taxotere) to compare the effectiveness of topical chemotherapy. The topical application of DTX HPV gel in tumor bearing mice resulted in nearly 4-fold tumor volume reduction which was equivalent to intravenous Taxotere therapy. Toxicity analysis of DTX HPV gel in comparison with intravenous Taxotere dosing showcased remarkably lower levels of toxicity biomarkers (aspartate transaminase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN), and creatinine), indicating improved safety of topical chemotherapy. Overall results warranted the effectiveness of topical DTX chemotherapy to reduce tumor burden with substantially reduced risk of systemic toxicities in breast cancer.

Exploration of docetaxel palmitate and its solid lipid nanoparticles as a novel option for alleviating the rising concern of multi-drug resistance.

Docetaxel (DTX), a widely prescribed anticancer agent, is now associated with increased instances of multidrug resistance. Also, being a problematic BCS class IV drug, it poses challenges for the formulators. Henceforth, it was envisioned to synthesize an analogue of DTX with a biocompatible lipid, i.e., palmitic acid. The in-silico studies (molecular docking and simulation) inferred lesser binding of docetaxel palmitate (DTX-PL) with P-gp vis-à-vis DTX and paclitaxel, indicating it to be a poor substrate for P-gp efflux. Solid lipid nanoparticles (SLNs) of the conjugate were prepared using various lipids, viz. palmitic acid, stearic acid, cetyl palmitate and glyceryl monostearate. The characterization studies for the nanocarrier were performed for the surface charge, drug payload, micromeritics, release pattern of drug and surface morphology. From the cytotoxicity assays on resistant MCF-7 cells, it was established that the new analogue offered substantially decreased IC50 to that of DTX. Further, apoptosis assay also corroborated the results obtained in IC50 determination wherein, SA-SLNs showed the highest apoptotic index than free DTX. The conjugate not only enhanced the solubility but also offered lower plasma protein binding and improved pharmacokinetic and pharmacodynamic effect for DTX loaded SA-SLNs in apt animal models, and lower affinity to P-gp efflux. The studies provide preliminary evidence and a ray of hope for a better candidate in its nano version for safer and effective cancer chemotherapy.