The T-cell-directed vaccine BNT162b4 encoding conserved non-spike antigens protects animals from severe SARS-CoV-2 infection.

T cell responses play an important role in protection against beta-coronavirus infections, including SARS-CoV-2, where they associate with decreased COVID-19 disease severity and duration. To enhance T cell immunity across epitopes infrequently altered in SARS-CoV-2 variants, we designed BNT162b4, an mRNA vaccine component that is intended to be combined with BNT162b2, the spike-protein-encoding vaccine. BNT162b4 encodes variant-conserved, immunogenic segments of the SARS-CoV-2 nucleocapsid, membrane, and ORF1ab proteins, targeting diverse HLA alleles. BNT162b4 elicits polyfunctional CD4+ and CD8+ T cell responses to diverse epitopes in animal models, alone or when co-administered with BNT162b2 while preserving spike-specific immunity. Importantly, we demonstrate that BNT162b4 protects hamsters from severe disease and reduces viral titers following challenge with viral variants. These data suggest that a combination of BNT162b2 and BNT162b4 could reduce COVID-19 disease severity and duration caused by circulating or future variants. BNT162b4 is currently being clinically evaluated in combination with the BA.4/BA.5 Omicron-updated bivalent BNT162b2 (NCT05541861).

Improved Oral Bioavailability and Gastrointestinal Stability of Amphotericin B through Fatty Acid Conjugation Approach.

Amphotericin B (AmB) is one of the most effective drugs used in the treatment of leishmaniasis and systemic fungal infections. Considering the global burden of leishmaniasis, ∼90% of disease cases occur in developing countries, suggestive of the need for an affordable AmB therapy. However, owing to the physicochemical properties of AmB, all the clinically available formulations must be administered by intravenous route, thereby creating a significant hurdle in patients' access to AmB due to pharmacoeconomic considerations. We have previously demonstrated that lipid conjugation (e.g., fatty acids) to AmB significantly decreases the toxicity of resulting prodrug by a favorable alteration in the aggregation pattern. The hypothesis of the present work was to investigate the potential of the previously established AmB-lipid conjugate [AmB-oleyl conjugate (AmB-OA)] in improving the physicochemical properties such as gastric instability and lower intestinal permeability that otherwise limits the oral delivery of AmB. The synthesized AmB-OA conjugate was remarkably stable at gastric pH in contrast to AmB and exhibited significantly higher permeation across the Caco-2 monolayer (indicative of intestinal permeability). Mechanistic studies revealed that AmB-OA retained an equivalent antifungal activity. Also, AmB-OA was found to interact preferentially with intracellular membranes of Saccharomyces cerevisiae, while AmB interacted with the plasma membrane. The results of Caco-2 monolayer permeation experiments were further confirmed by in vivo pharmacokinetics, which showed that AmB-OA exhibited a 3.13-fold increase in the Cmax and a 4.88-fold increase in AUCTot as compared to AmB. In conclusion, the lipid conjugation approach may provide an effective solution for current challenges in designing drug delivery systems intended for oral AmB therapy.

Application of a Quality-By-Design Approach to Optimise Lipid-Polymer Hybrid Nanoparticles Loaded with a Splice-Correction Antisense Oligonucleotide: Maximising Loading and Intracellular Delivery.

BACKGROUND: Antisense oligonucleotides (ASOs) are promising therapeutics for specific modulation of cellular RNA function. However, ASO efficacy is compromised by inefficient intracellular delivery. Lipid-polymer hybrid nanoparticles (LPNs) are attractive mediators of intracellular ASO delivery due to favorable colloidal stability and sustained release properties. METHODS: LPNs composed of cationic lipidoid 5 (L5) and poly(DL-lactic-co-glycolic acid) were studied for delivery of an ASO mediating splice correction of a luciferase gene transcript (Luc-ASO). Specific purposes were: (i) to increase the mechanistic understanding of factors determining the loading of ASO in LPNs, and (ii) to optimise the LPNs and customise them for Luc-ASO delivery in HeLa pLuc/705 cells containing an aberrant luciferase gene by using a quality-by-design approach. Critical formulation variables were linked to critical quality attributes (CQAs) using risk assessment and design of experiments, followed by delineation of an optimal operating space (OOS). RESULTS: A series of CQAs were identified based on the quality target product profile. The L5 content and L5:Luc-ASO ratio (w/w) were determined as critical formulation variables, which were optimised systematically. The optimised Luc-ASO-loaded LPNs, defined from the OOS, displayed high loading and mediated splice correction at well-tolerated, lower doses as compared to those required for reference L5-based lipoplexes, L5-modified stable nucleic acid lipid nanoparticles or LPNs modified with dioleoyltrimethylammonium propane (conventional cationic lipid). CONCLUSIONS: The optimal Luc-ASO-loaded LPNs represent a robust formulation that mediates efficient intracellular delivery of Luc-ASO. This opens new avenues for further development of LPNs as a broadly applicable technology platform for delivering nucleic acid cargos intracellularly.

Identification of Factors of Importance for Spray Drying of Small Interfering RNA-Loaded Lipidoid-Polymer Hybrid Nanoparticles for Inhalation.

BACKGROUND: With the recent approval of the first small interfering RNA (siRNA) therapeutic formulated as nanoparticles, there is increased incentive for establishing the factors of importance for the design of stable solid dosage forms of such complex nanomedicines. METHODS: The aims of this study were: (i) to identify factors of importance for the design of spray-dried siRNA-loaded lipidoid-poly(DL-lactic-co-glycolic acid) hybrid nanoparticles (LPNs), and (ii) to evaluate their influence on the resulting powders by using a quality-by-design approach. Critical formulation and process parameters were linked to critical quality attributes (CQAs) using design of experiments, and an optimal operating space (OOS) was identified. RESULTS: A series of CQAs were identified based on the quality target product profile. The loading (ratio of LPNs to the total solid content) and the feedstock concentration were determined as critical parameters, which were optimized systematically. Mannitol was chosen as stabilizing excipient due to the low water content of the resulting powders. The loading negatively affected the colloidal stability of the LPNs, whereas feedstock concentration correlated positively with the powder particle size. The optimal mannitol-based solid formulation, defined from the OOS, displayed a loading of 5% (w/w), mass median aerodynamic diameter of 3.3 ± 0.2 µm, yield of 60.6 ± 6.6%, and a size ratio of 1.15 ± 0.03. Dispersed micro-embedded LPNs had preserved physicochemical characteristics as well as in vitro siRNA release profile and gene silencing, as compared to non-spray-dried LPNs. CONCLUSION: The optimal solid dosage forms represent robust formulations suitable for higher scale-up manufacturing.

Design of Inhalable Solid Dosage Forms of Budesonide and Theophylline for Pulmonary Combination Therapy.

Corticosteroid resistance poses a major challenge to effective treatment of chronic obstructive pulmonary diseases. However, corticosteroid resistance can be overcome by co-administration of theophylline. The aim of this study was to formulate the corticosteroid budesonide with theophylline into inhalable dry powders intended for pulmonary combination therapy. Four types of spray-dried powders were prepared: (i) budesonide and theophylline co-dissolved and processed using a 2-fluid nozzle spray drier, (ii) budesonide nanocrystals and dissolved theophylline co-dispersed and processed using a 2-fluid nozzle spray drier, (iii) dissolved budesonide and dissolved theophylline processed using a 3-fluid nozzle spray drier, and (iv) budesonide nanocrystals and dissolved theophylline processed using a 3-fluid nozzle spray drier. Spray drying from the solutions resulted in co-amorphous (i) and partially amorphous powders (iii), whereas spray drying of the nanosuspensions resulted in crystalline products (ii and iv). Even though budesonide was amorphous in (i) and (iii), it failed to exhibit any dissolution advantage over the unprocessed budesonide. In contrast, the dissolution of budesonide from its nanocrystalline formulations, i.e., (ii) and (iv), was significantly higher compared to a physical mixture or unprocessed budesonide. Furthermore, the spray-dried powders obtained from the 2-fluid nozzle spray drier, i.e., (i) and (ii), exhibited co-deposition of budesonide and theophylline at the same weight ratio in the aerodynamic assessment using the New Generation Impactor. In contrast, the depositions of budesonide and theophylline deviated from the starting weight ratio in the aerodynamic assessment of spray-dried powders obtained from the 3-fluid nozzle spray drier, i.e., (iii) and (iv). Based on these results, the powders spray-dried from the suspension by using the 2-fluid nozzle spray drier, i.e., (ii), offered the best formulation properties given the physically stable crystalline solid-state properties and the co-deposition profile.

Formulating Inhalable Dry Powders Using Two-Fluid and Three-Fluid Nozzle Spray Drying.

PURPOSE: The spray drying process is widely applied for pharmaceutical particle engineering. The purpose of this study was to investigate advantages and disadvantages of two-fluid nozzle and three-fluid nozzle spray drying processes to formulate inhalable dry powders. METHODS: Budesonide nanocomposite microparticles (BNMs) were prepared by co-spray drying of budesonide nanocrystals suspended in an aqueous mannitol solution by using a two-fluid nozzle spray drying process. Budesonide-mannitol microparticles (BMMs) were prepared by concomitant spray drying of a budesonide solution and an aqueous mannitol solution using a spray drier equipped with a three-fluid nozzle. The resulting dry powders were characterized by using X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), dynamic mechanical analysis (DMA) and Raman microscopy. A Next Generation Impactor was used to evaluate the aerodynamic performance of the dry powders. RESULTS: XRPD and DMA results showed that budesonide remained crystalline in the BNMs, whereas budesonide was amorphous in the BMMs. Spray drying of mannitol into microparticles resulted in a crystalline transformation of mannitol, evident from XRPD, DSC and Raman spectroscopy analyses. Both BMMs and BNMs displayed a faster dissolution rate than bulk budesonide. The yield of BNMs was higher than that of BMMs. The mass ratio between budesonide and mannitol was preserved in the BNMs, whereas the mass ratio in the BMMs was higher than the theoretical ratio. CONCLUSIONS: Spray drying is an enabling technique for preparation of budesonide amorphous solid dispersions and nanocrystal-embedded microparticles. Two-fluid nozzle spray drying is superior to three-fluid nozzle spray drying in terms of yield.

Triple antioxidant SNEDDS formulation with enhanced oral bioavailability: Implication of chemoprevention of breast cancer.

The present study aimed to develop quercetin, resveratrol and genistein loaded self-nanoemulsifying drug delivery system (SNEDDS) by QbD approach in order to improve their oral bioavailability and antioxidant potential. The size and PDI of the optimized formulation were found to be <200nm and <0.3, respectively. DPPH scavenging assay showed comparable antioxidant activity of antioxidant loaded SNEDDS to free antioxidants combination. Furthermore, coumarin-6 loaded SNEDDS formulation showed rapid internalization within 1h of incubation by Caco-2 cells. Moreover, the pharmacokinetic studies in rats for the optimized formulation and free antioxidant suspension were performed. SNEDDS have significantly increased the Cmax and area under curve (AUC) of all three antioxidants. The SNEDDS demonstrated ~4.27 fold enhancement in oral bioavailability of quercetin, ~1.5 fold in case of resveratrol and ~2.8 fold in case of genistein as compared to free antioxidants suspension. Finally, the prophylactic antitumor efficacy of developed formulation was tested against DMBA induced breast cancer model in rats, which demonstrated enhanced abeyance towards the tumor growth as compared to free antioxidants.

Enhanced Antitumor Efficacy and Reduced Toxicity of Docetaxel Loaded Estradiol Functionalized Stealth Polymeric Nanoparticles.

In spite of extensive research over the decades, breast cancer treatment is still a major challenge due to nonspecific distribution of the chemotherapeutics. This void can be filled by restricting the distribution of chemotherapeutics toward the cancerous cells. In the present report estradiol (E2) functionalization of docetaxel (DTX) loaded PLGA nanoparticles was supposed to have specific distribution of DTX to cancerous cells simultaneously avoiding the nonspecific distribution toward the normal cells. In line, E2-PEG-PLGA conjugate was synthesized and characterized by FTIR and NMR spectroscopy. Extensive optimization of different process variables resulted in the formation of spherical E2-PEG-PLGA NPs in the size range of 228.5 ± 11.8 nm and entrapment efficiency of 94.25 ± 2.49. Trehalose (5% w/v) resulted in the formation of a fluffy, easy to redisperse freeze-dried cake of nanoparticles. PXRD analysis revealed the amorphous nature of DTX encapsulated within the nanoparticles. X-ray photoelectron spectroscopy confirmed the presence of E2 over the surface of nanoparticles. In line with our hypothesis, cellular uptake studies on ER positive MCF-7 cells revealed relatively higher uptake and efficient localization into the nuclear region of E2-PEG-PLGA NPs in comparison with plain counterparts, while in the case of ER negative HeLa cells E2-PEG-PLGA NPs showed no difference in fluorescence pattern as compared to MCF-7 cells incubated with unmodified nanoformulation, indicating nonspecific delivery of DTX. Moreover, MTT assay revealed relatively higher cytotoxicity of E2-PEG-PLGA NPs in comparison with free DTX. Furthermore, in vivo pharmacokinetic studies revealed 9.36- and 4.79-fold enhancement in circulation half-life and AUC(0-∞), respectively, of E2-PEG-PLGA NPs in comparison with Taxotere. In vivo antitumor efficacy in DMBA induced rat model demonstrated significant reduction in tumor volume in comparison with the plain counterpart (PLGA-NPs) and a marketed formulation, Taxotere. Moreover, the safety of the estradiol functionalized PLGA NPs was confirmed by hepato- and nephrotoxicity studies.

Oral bioavailability and pharmacodynamic activity of hesperetin nanocrystals generated using a novel bottom-up technology.

In the present study, nanocrystalline solid dispersion (NSD) was developed to enhance the release rate and oral bioavailability of hesperetin (HRN). NSD of HRN was prepared using a novel bottom-up technology platform. It is a spray drying based technology to generate solid particles, containing drug nanocrystals dispersed in small molecule excipients. HRN and mannitol were used in a 5:5 ratio, and an average crystallite size of HRN in NSD with mannitol was found to be 137.3 ± 90.0 nm. An in vitro release study revealed a statistically significant release rate enhancement for HRN nanocrystals (46.3 µg/mL/min) as compared to that of the control (29.5 µg/mL/min). Further, a comparative oral bioavailability study of NSD and control in Sprague-Dawley rats established significant improvement in Cmax and oral bioavailability (AUC0-∞) by 1.79- and 2.25-fold, respectively, for HRN nanocrystals. The findings of oral bioavailability were corroborated by intestinal uptake and Caco-2 cell uptake studies, wherein HRN, when administered in nanocrystalline form, showed higher penetration in intestinal mucosa and higher uptake in Caco-2 cells. Finally, the therapeutic efficacy of HRN nanocrystals was tested by a reactive oxygen species (ROS) generation assay and carrageenan induced anti-inflammatory model. HRN nanocrystals markedly inhibited ROS generation in MCF-7 cells, and carrageenan induced inflammation in rats. The process of NSD formation was found to be based on classical nucleation theory wherein mannitol contributed to NSD formation by acting as a plasticizer and crystallization inducer, and by providing sites for heterogeneous nucleation/crystallization.

Phytantriol Based "Stealth" Lyotropic Liquid Crystalline Nanoparticles for Improved Antitumor Efficacy and Reduced Toxicity of Docetaxel.

PURPOSE: The present work focuses on design and development of surface functionalized LCNPs for improving tumor delivery of DTX. METHODS: Phytantriol based "stealth" LCNPs were prepared using hydrotrope method and optimized. The potential of developed formulation was further assessed using cell culture experiments, in vivo pharmacokinetics, in vivo pharmacodynamics and toxicity studies. RESULTS: A biphasic drug release pattern was observed with sustained release of drug till 72 h. In vitro cell culture experiments revealed efficient internalization within MCF-7 cells with 25.80-fold decrease in IC50 value for PEG-LCNPs as compared to free DTX. Mechanistic insights demonstrated preferential co-localization of LCNPs in the vicinity of the nucleus. Furthermore, in vivo pharmacokinetic studies revealed 14.45-fold enhancement in circulation half-life of PEG-LCNPs as compared to marketed formulation Taxotere®. In vivo efficacy studies PEG-LCNPs in DMBA induced breast cancer model revealed ~81% reduction in the tumor burden compared to Taxotere® which caused/achieve only 47% reduction or showed only 47% decrease. Furthermore, safety profile was noted for PEG-LCNPs as compared to Taxotere®, measured as a function of hepato- and nephro-toxicity. CONCLUSIONS: Surface functionalization of LCNPsis a viable approach for improving the therapeutic potential of DTX.

Macromolecular bipill of gemcitabine and methotrexate facilitates tumor-specific dual drug therapy with higher benefit-to-risk ratio.

The present study reports the synthesis, characterization, and biological evaluation of a novel macromolecular bipill, synthesized by appending two different anticancer agents, viz., gemcitabine (GEM) and methotrexate (MTX), to the distal ends of a long-circulating poly(ethylene glycol) (PEG) spacer. Covalent conjugation of GEM and MTX via PEG linker not only transformed the solubility profiles of constituent drug molecules, but significantly improved their stability in the presence of plasma. In vitro cytotoxicity studies confirmed that GEM-PEG-MTX exerts higher cytotoxicity (IC50 0.181 µM at 24 h) in human breast adenocarcinoma MCF-7 cell lines, when compared to free drug congeners, i.e., free GEM (IC50 0.294 µM at 24 h) and free MTX (IC50 0.591 µM at 24 h). Tumor growth inhibition studies in chemically induced breast cancer bearing rats established the superiority of GEM-PEG-MTX conjugate over all other pharmaceutical preparations including free drugs, physical mixture of GEM and MTX, and PEGylated GEM/MTX. Toxicity studies in tumor bearing rats as well as healthy mice corroborated that dual drug conjugation is an effective means to synergize the therapeutic indices of potential drug candidates while alleviating drug-associated side effects.

Lyotropic liquid crystalline nanoparticles of CoQ10: implication of lipase digestibility on oral bioavailability, in vivo antioxidant activity, and in vitro-in vivo relationships.

The present investigation reports implications of the lipase digestibility of lyotropic liquid crystalline nanoparticles (LCNPs) on the oral bioavailability, in vivo antioxidant potential, and in vitro-in vivo relationship (IVIVR) of CoQ10 loaded LCNPs prepared from glyceryl monooleate (GLCQ) and phytantriol (PLCQ). Exhaustive optimization of the process variables was carried out, and optimized lyophilized formulations were found to have particle sizes of 140.45 ± 5.47 nm and 238.42 ± 8.35 nm and a polydispersity index (PDI) of 0.15 ± 0.01 and 0.22 ± 0.03 for GLCQ and PLCQ, respectively. The entrapment efficiency at 10% theoretical loading was found to be >90% in both the cases. The morphological characteristics of the developed formulations were assessed using high resolution transmission electron microscopy and small-angle X-ray scattering analysis, which showed hexagonal (HII) structure. The developed formulations were also found to be stable in simulated gastrointestinal fluids for the stipulated period of time. The in vitro drug release studies revealed a bimodal sustained release drug profile with Higuchi type release kinetics as the best fit release model for both the formulations. The best fit release models were found to be of the Hixson Crowell type in the case of GLCQ when carried out in lipase rich media, suggestive of matrix erosion and subsequent formation of secondary structures, which was further corroborated by carrier degradation studies. Furthermore, 9.1- and 10.67-fold increase in Caco-2 cell uptake was observed in the case of GLCQ and PLCQ, respectively, attributed to the formation of the virtual channel pathway as a probable absorption mechanism. Consequently, 7.09- and 8.67-fold increase in oral bioavailability was observed in the case of GLCQ and PLCQ, respectively. The IVIVR was also established with r(2) values in the order of 0.996 and 0.999 for GLCQ and PLCQ, respectively, in contrast to that of 0.484 for free CoQ10. Finally, in vivo prophylactic antioxidant efficacy against the STZ-treated rats using various markers such as GSH, LDH, SOD, MDA, glucose level, and body weight showed significantly higher antioxidant activity of CoQ10-LCNPs as compared to that of free CoQ10. In a nutshell, the developed formulation strategy poses great potential in improving the oral bioavailability of difficult-to-deliver drugs such as CoQ10.

Improved stability and antidiabetic potential of insulin containing folic acid functionalized polymer stabilized multilayered liposomes following oral administration.

The present study reports the folic acid (FA) functionalized insulin loaded stable liposomes with improved bioavailability following oral administration. Liposomes were stabilized by alternating coating of negatively charged poly(acrylic acid) (PAA) and positively charged poly(allyl amine) hydrochloride (PAH) over liposomes. Furthermore, folic acid was appended as targeting ligand by synthesizing folic acid-poly(allyl amine) hydrochloride conjugate. The insulin entrapped within the freeze-dried formulation was found stable both chemically as well as conformationally and developed formulation exhibited excellent stability in simulated biological fluids. Caco-2 cell and ex vivo intestinal uptake studies revealed higher uptake of folic acid functionalized layersomes in comparison with their plain counterparts. In vivo pharmacodynamic and pharmacokinetic studies further revealed almost double hypoglycemia and approximately 20% relative bioavailability in comparison with subcutaneously administered standard insulin solution. Overall the proposed strategy is expected to contribute significantly in the field of designing ligand-anchored, polyelectrolyte-based stable systems in drug delivery.

Bicontinuous cubic liquid crystalline nanoparticles for oral delivery of Doxorubicin: implications on bioavailability, therapeutic efficacy, and cardiotoxicity.

PURPOSE: The present study explores the potential of bicontinous cubic liquid crystalline nanoparticles (LCNPs) for improving therapeutic potential of doxorubicin. METHODS: Phytantriol based Dox-LCNPs were prepared using hydrotrope method, optimized for various formulation components, process variables and lyophilized. Structural elucidation of the reconstituted formulation was performed using HR-TEM and SAXS analysis. The developed formulation was subjected to exhaustive cell culture experiments for delivery potential (Caco-2 cells) and efficacy (MCF-7 cells). Finally, in vivo pharmacokinetics, pharmacodynamic studies in DMBA induced breast cancer model and cardiotoxicity were also evaluated. RESULTS: The reconstituted formulation exhibited Pn3m type cubic structure, evident by SAXS and posed stability in simulated gastrointestinal fluids and at accelerated stability conditions for 6 months. Dox-LCNPs revealed significantly higher cell cytotoxicity (16.23-fold) against MCF-7 cell lines as compared to free drug owing to its preferential localization in the vicinity of nucleus. Furthermore, Caco-2 cell experiments revealed formation of reversible "virtual pathways" in the cell membrane for Dox-LCNPs and hence posed significantly higher relative oral bioavailability (17.74-fold). Subsequently, Single dose of Dox-LCNPs (per oral) led to significant reduction in % tumor burden (~42%) as compared that of ~31% observed in case of Adriamycin® (i.v.) when evaluated in DMBA induced breast cancer model. Moreover, Dox induced cardiotoxicity was also found to be significantly lower in case of Dox-LCNPs as compared to clinical formulations (Adriamycin® and Lipodox®). CONCLUSION: Incorporation of Dox in the novel LCNPs demonstrated improved antitumor efficacy and safety profile and can be a viable option for oral chemotherapy.

Solidified self-nanoemulsifying formulation for oral delivery of combinatorial therapeutic regimen: part II in vivo pharmacokinetics, antitumor efficacy and hepatotoxicity.

PURPOSE: The present work focuses on the in vivo evaluation of tamoxifen and quercetin combination loaded into solid self-nanoemulsifying drug delivery system (s-Tmx-QT-SNEDDS). METHODS: Lyophilization was employed to prepare s-Tmx-QT-SNEDDS using Aerosil 200 as carrier. The developed formulation was evaluated for in vitro cell cytotoxicity, in vivo pharmacokinetics, antitumor efficacy and toxicity studies. RESULTS: In vivo pharmacokinetics revealed ~8-fold and ~4-fold increase in oral bioavailability of tamoxifen and quercetin, respectively as compared to free counterparts. s-Tmx-QT-SNEDDS exhibited significantly higher cell cytotoxicity, as compared to free drug combination revealing ~32-fold and ~22-fold higher dose reduction index for tamoxifen and quercetin, respectively estimated using median effect dose analysis. s-Tmx-QT-SNEDDS could suppress tumor growth in DMBA induced tumor bearing animals by ~80% in contrast to ~35% observed with tamoxifen citrate. The significant appreciation in antitumor efficacy was further supported by normalized levels of tumor angiogenesis markers (MMP-2 and MMP-9). Finally, complete obliteration in tamoxifen induced hepatotoxicity was observed upon administration of developed formulation in contrast to that of clinically available tamoxifen citrate when measured as function of hepatotoxicity markers and histopathological changes. CONCLUSIONS: In nutshell, co-encapsulation of quercetin with tamoxifen in solid SNEDDS poses great potential in improving the therapeutic efficacy and safety of tamoxifen.

Solidified self-nanoemulsifying formulation for oral delivery of combinatorial therapeutic regimen: part I. Formulation development, statistical optimization, and in vitro characterization.

PURPOSE: The present work reports rationalized development and characterization of solidified self-nanoemulsifying drug delivery system for oral delivery of combinatorial (tamoxifen and quercetin) therapeutic regimen. METHODS: Suitable oil for the preparation of liquid SNEDDS was selected based on the maximum saturation solubility of both the drugs while surfactant and co-surfactant were selected based on their emulsification ability. Extreme vertices mixture design and 3(2) full factorial design were implemented for optimization of liquid SNEDDS and concentration of solid carrier in lyophilization mixture. Finally, extensive characterization of the developed formulation was performed and in vitro cellular uptake was evaluated in Caco-2 cell culture model. RESULTS: Extreme vertices mixture design indicated the desirability of 0.663, corresponded to 40:30:30 w/w as optimum ratio of oil (Capmul® MCM), surfactant (Cremophor RH 40) and co-surfactant (Labrafil 1944CS) in liquid SNEDDS, which solubilized high amount of tamoxifen (10 mg/g) and quercetin (19.44 mg/g). A, 3(2) full factorial design revealed the optimum concentration of the selected solid carrier (Aerosil 200) of 5.24% w/w and 1.61, when measured in terms of total solid content and liquid SNEDDS: Aerosil 200 ratio, respectively. The developed formulation revealed instantaneous emulsification (in < 2 min), while maintaning all the quality attributes even after storage at accelerated stability condition for 6 months. Finally, the developed formulation revealed 9.63-fold and 8.44-fold higher Caco-2 uptake of tamoxifen and quercetin, respectively in comparison with free drug counterparts. CONCLUSIONS: The developed formulation strategy revealed a great potential for oral delivery of combination drugs having utmost clinical relevance.

Enhanced antitumor efficacy and counterfeited cardiotoxicity of combinatorial oral therapy using Doxorubicin- and Coenzyme Q10-liquid crystalline nanoparticles in comparison with intravenous Adriamycin.

Present study focuses on enhancing oral antitumor efficacy and safety of Dox-LCNPs in combination with CoQ10-LCNPs. Drug-loaded-LCNPs were prepared by solvent-diffusion-evaporation method and optimized. Median effect analysis suggested dose-reduction-index of 16.84- and 5.047-fold and strong synergism for combination at 1:10 dose ratio owing to higher cellular uptake, nuclear colocalization, higher apoptotic index and 8-OHdG levels. The prophylactic antitumor efficacy of the CoQ10-LCNPs was also established using tumor induction and progression studies. Finally, therapeutic antitumor efficacy was found to be significantly higher (~1.76- and ~4.5-fold) for the combination as compared to Dox-LCNPs (per oral) and Adriamycin (i.v.) respectively. Notably, level of residual tumor burden was insignificant (P>0.05) after 30days in case of combination and LipoDox® (i.v.). Interestingly, with Dox-induced-cardiotoxicity was completely counterfeited in combination. In nutshell, LCNPs pose great potential in improving the therapeutic efficacy of drugs by oral route of administration. FROM THE CLINICAL EDITOR: This study describes the use of liquid crystalline nanoparticles containing coenzyme Q10 and doxorubicin. The nano-conjugates not only provided an enhanced oral treatment option for a tumor model, but prevented cardiotoxicity, a major complication of this drug when delivered via conventional methods.

Novel self-nanoemulsifying formulation of quercetin: Implications of pro-oxidant activity on the anticancer efficacy.

The present work focuses on the anticancer potential of quercetin (QT) loaded self-nanoemulsifying drug delivery system (QT-SNEDDS) composed of Capmul MCM, Tween 20 and ethanol. In vitro cell culture studies revealed potential cell cytotoxicity of developed formulation mediated by its ability to induce DNA damage and apoptosis in MCF-7 cells. QT-SNEDDS at a dose of 50mg/kg demonstrated the antioxidant activity measured as function of prophylactic antitumor efficacy against DMBA induced breast tumors which revealed higher latency to the tumor growth as compared to free QT. This appreciation was further supported by normalized levels of tumor angiogenesis markers (MMP-2, MMP-9, TNF-α and IL-6). At higher doses (100mg/kg) the pro-oxidant activity was noted and exhibited significantly higher therapeutic anticancer efficacy (~65% tumor suppression) in the same model as compared to that of free QT (~20%). Finally, safety profile of developed formulation was established assessing various hepatotoxicity markers. FROM THE CLINICAL EDITOR: This basic science study focuses on the anticancer potential of a specific quercetin loaded self-nanoemulsifying drug delivery system. At higher doses significantly higher therapeutic anticancer efficacy (~65% tumor suppression) was noted in the same model as compared to that of free quercetin (~20%).

Co-encapsulation of tamoxifen and quercetin in polymeric nanoparticles: implications on oral bioavailability, antitumor efficacy, and drug-induced toxicity.

The present investigation reports the preparation, optimization, and characterization of orally administrable PLGA-NPs co-encapsulated with tamoxifen (Tmx) and quercetin (QT). The developed formulation was found to have particle size 185.3 ± 1.20 nm, PDI 0.184 ± 0.004, entrapment efficiency 67.16 ± 1.24% Tmx, 68.60 ± 1.58% QT at a Tmx/QT ratio of 1:2 w/w. The stability of the freeze-dried formulation was established in simulated gastrointestinal fluids for 8 h and at accelerated stability condition for 3 months. DPPH free radical scavenging assay confirmed that the functional architecture of QT was retained in freeze-dried NPs. Higher cellular uptake, cytotoxicity, and nuclear co-localization of Tmx-QT-NPs in MCF-7 cells revealed higher efficiency of the formulation. At the same time, higher Caco-2 cell uptake revealed its potential for oral delivery, which was well corroborated with in vivo pharmacokinetics, which suggested ∼ 5-fold and ∼ 3-fold increase in oral bioavailability as compared to the free Tmx citrate and free QT, respectively. Concomitantly, significantly higher tumor suppression was observed in the case of the developed formulation in contrast to respective free drug(s) and their combination when tested against a DMBA-induced breast cancer model in female SD rats. Multiple oral administrations of Tmx-QT-NPs efficiently controlled the tumor angiogenesis as revealed by normalized levels of respective markers (MMP-2 and MMP-9). The safety profile of Tmx-QT-NPs was also established, and no measurable hepatotoxicity or oxidative stress was observed when measured as a function of respective biochemical markers in contrast to free drug(s) and their combinations. In a nutshell, the co-encapsulation strategy with PLGA-NPs could be a promising approach in improving oral delivery of Tmx and QT for cancer therapy.

Enhanced transfection efficiency and reduced cytotoxicity of novel lipid-polymer hybrid nanoplexes.

The present study reports the development, characterization, and evaluation of novel polyelectrolytes stabilized lipoplexes as a nonviral vector for gene delivery. In order to achieve the advantage of both DOTAP (1,2-dioleoyl-3-trimethylammonium propane) and PEI (high transfection efficiency) a system was hypothesized in which DOTAP/phosphatidyl choline (PC) lipoplexes were electrostatically coated with anionic poly(acrylic acid) (PAA) and cationic polyethylenimine (PEI) alternatively to finally shape a robust structure PEI-PAA-DOTAP/PC-lipoplexes (nanoplexes). The nanoplexes were found to have size of 242.6 ± 9.4 nm and zeta potential of +23.1 ± 1.5 mV. Following development nanoplexes were evaluated for cellular uptake, nuclear colocalization, transfection efficiency, and cellular toxicity in MCF-7, HeLa, and HEK-293 cell lines. In support of our hypothesis nanoplexes exhibited higher uptake and nuclear colocalization in comparison with DOTAP/PC, DOTAP/DOPE lipoplexes, and PEI polyplexes. Nanoplexes also exhibited 50-80, 11-12, 6-7, and 5-6 fold higher transfection efficiency in comparison with DOTAP/PC-lipoplexes, DOTAP/DOPE-lipoplexes, PEI-polyplexes, and lipofectamine, respectively, and significantly lower toxicity in comparison with DOTAP/PC, DOTAP/DOPE lipoplexes, PEI polyplexes, and commercial lipofectamine.