Recent Advancement and Technical Challenges in Developing Small Extracellular Vesicles for Cancer Drug Delivery.

Extracellular vesicles (EVs) are a heterogeneous population of lipid bilayer membrane-enclosed vesicles and act like 'messages in a bottle' in cell-cell communication by transporting their cargoes to recipient cells. Small EVs (sEVs, < 200 nm) are highly researched recently and have been harnessed as novel delivery systems for the treatment of various diseases, including neurodegenerative disorders, cardiovascular diseases, and most importantly cancer primarily because of their non-immunogenicity, tissue penetration and cell-tropism. This review will first provide a comprehensive overview of sEVs regarding the current understanding on their properties, biogenesis, new classification by the ISEV, composition, as well as their roles in cancer development (thereby called "oncosomes"). The primary focus will be given to the current state of sEVs as natural nanocarriers for cancer drug delivery, the technologies and challenges involved in sEV isolation and characterization, therapeutic cargo loading, and surface modification to enhance tumor-targeting. We will also provide examples of sEV products under clinical trials. Furthermore, the current challenges as well as the advance in "sEV mimetics" to address some of the sEVs limitations is briefly discussed. We seek to advance our understanding of sEVs to unlock their full potential as superior drug delivery vehicles in cancer therapy.

Dual Activity of Hydroxypropyl-ß-Cyclodextrin and Water-Soluble Carriers on the Solubility of Carvedilol.

Carvedilol (CAR) is a non-selective α and ß blocker categorized as class II drug with low water solubility. Several recent studies have investigated ways to overcome this problem. The aim of the present study was to combine two of these methods: the inclusion complex using hydroxypropyl-ß-cyclodextrin (HPßCD) with solid dispersion using two carriers: Poloxamer 188 (PLX) and Polyvinylpyrrolidone K-30 (PVP) to enhance the solubility, bioavailability, and the stability of CAR. Kneading method was used to prepare CAR-HPßCD inclusion complex (KD). The action of different carriers separately and in combination on Carvedilol solubility was investigated in three series. CAR-carrier and KD-carrier solid dispersions were prepared by solvent evaporation method. In vitro dissolution test was conducted in three different media: double-distilled water (DDW), simulative gastric fluid (SGF), and PBS pH 6.8 (PBS). The interactions between CAR, HPßCD, and different carriers were explored by Fourier transform infrared spectroscopy (FTIR), powder X-ray diffractometry (XRD), and differential scanning colorimetry (DSC). The results showed higher solubility of CAR in KD-PVP solid dispersions up to 70, 25, and 22 fold compared to pure CAR in DDW, SGF, and PBS, respectively. DSC and XRD analyses indicated an improved degree of transformation of CAR in KD-PVP solid dispersion from crystalline to amorphous state. This study provides a new successful combination of two polymers with the dual action of HPßCD and PLX/PVP on water solubility and bioavailability of CAR.

Functionalisation of extracellular vesicles with cyclic-RGDyC potentially for glioblastoma targeted intracellular drug delivery.

With the intrinsic ability to cross the blood-brain barrier, small extracellular vesicles (sEVs) hold promise as endogenous brain-targeted drug delivery nano-platforms for glioblastoma (GBM) treatment. To increase GBM targetability, this study aimed to functionalise sEVs with cyclic arginine-glycine-aspartic acid-tyrosine-cysteine (cRGDyC), a ligand for integrin (αvß3) that is overexpressed in GBM cells. Firstly, the intrinsic cellular uptake of sEVs derived from GBM U87 and pancreatic cancer MIA PaCa-2 cells was investigated on the donor cells. To obtain functionalised sEVs (cRGDyC-sEVs), DSPE-mPEG2000-maleimide was incubated with the selected (U87) sEVs, and cRGDyC was subsequently conjugated to the maleimide groups via a thiol-maleimide coupling reaction. The GBM cell targetability and intracellular trafficking of cRGDyC-sEVs were evaluated on U87 cells by fluorescence and confocal microscopy, using unmodified sEVs as a reference. The cytotoxicity of doxorubicin-loaded vesicles (Dox@sEVs, Dox@cRGDyC-sEVs) was compared with a standard liposome formulation (Dox@Liposomes) and free Dox. Both U87 and MIA PaCa-2 cell-derived sEVs displayed tropism with the former being >4.9-fold more efficient to be internalised into U87. Therefore, the U87-derived sEVs were chosen for GBM-targeting. Approximately 4000 DSPE-mPEG2000-maleimide were inserted onto each sEV with cRGDyC conjugated to the maleimide group. The cell targetability of cRGDyC-sEVs to U87 cells improved 2.4-fold than natural sEVs. Despite their proneness to be colocalised with endosomes/lysosomes, both Dox@sEVs and Dox@cRGDyC-sEVs showed superior cytotoxicity to U87 GBM cells compared to Dox@Liposomes, particularly Dox@cRGDyC-sEVs. Overall, U87-derived sEVs were successufully conjugated with cRGDyC via a PEG linker, and cRGDyC-sEVs were demonstrated to be a potnetial integrin-targeting drug delivery vehicle for GBM treatment. Graphic abstract.

A simple approach to re-engineering small extracellular vesicles to circumvent endosome entrapment.

Small extracellular vesicles (sEVs) have emerged as attractive drug delivery systems. However, the intracellular release of their cargoes is restricted. This study aimed to develop an efficient approach to re-engineer sEVs by hybridisation with pH-sensitive liposomes (PSLs) and investigate their endosome escape potential. MIA PaCa-2 cell-derived sEVs and PSLs were fused via three methods, and fusion efficiency (FE) was measured using a fluorescence resonance energy transfer assay and nanoparticle tracking analysis. Cellular uptake, intracellular trafficking, and cytotoxicity of doxorubicin-loaded vesicles (Dox@hybrids, Dox@sEVs, and Dox@PSLs) were investigated on MIA PaCa-2 cells. Among the three methods, Ca2+-mediated fusion was the simplest and led to a comparable FE with freeze-thaw method, which was significantly higher than PEG8000-mediated fusion. sEVs were more stable after hybridisation with PSLs. Confocal microscopy revealed that the hybrids internalised more efficiently than natural sEVs. While the internalised Dox@sEVs were primarily co-localised with endo/lysosomes even after 8 h, Dox from Dox@hybrids was found to escape from endosomes by 2 h and homogenously distributed in the cytosol before accumulated at nucleus, corresponding to the in vitro pH-responsive release profile. Consequently, Dox@hybrids enhanced cytotoxicity compared with Dox@sEVs, Dox@PSLs, or free drugs. Overall, the biomimetic nanosystem generated by simple Ca2+-mediated fusion was more stable and demonstrated higher efficiencies of cellular uptake and endosome escape compared to natural sEVs.

Cationic chitosan-modified silica nanoparticles for oral delivery of protein vaccine.

Mesoporous silica nanoparticles coated with Chitosan are exploited here as a potential carrier for oral vaccine delivery. Bovine serum albumin (BSA) was used as a protein antigen model to reveal the carrier property. Chitosan-coated BSA-loaded silica NPs had particle size 345 ± 60 nm with a cationic surface charge of 18.28 ± 0.71 mV. The encapsulation efficiency, drug loading was 25.34 ± 0.76 and 20.21 ± 0.48%, respectively. Transmission electron microscopy investigation showed the spherical shape of NPs, also confirmed surface coating around modified nanoparticles (NPs), and nitrogen absorption/desorption isotherm confirmed mesostructured inside the NPs. Fourier transform infrared spectroscopy did not show any physiochemical interactions between excipients and formulations. The structural stability of antigen after release from NPs was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, and chitosan-coated silica NPs exhibited a slow-release pattern. The results of in vivo experiments presented that chitosan-mesoporous silica NPs could induce a robust immune response in mice, indicating that chitosan-mesoporous silica NPs might be used as a promising carrier for oral vaccine delivery.

Novel scheme for rapid synthesis of hollow mesoporous silica nanoparticles (HMSNs) and their application as an efficient delivery carrier for oral bioavailability improvement of poorly water-soluble BCS type II drugs.

Hollow mesoporous silica nanoparticles (HMSNs) are one of the most promising carriers for drug delivery. However, a facile method to synthesize HMSNs has hardly been reported so far. The primary objective of our current study was to develop HMSNs using a simple, quick, and inexpensive method and evaluate their ability to enhance solubility, dissolution rate, and bioavailability of poorly water-soluble model BSC type II drug Carvedilol. Traditional mesoporous silica nanoparticles (MSNs) are synthesized using classical Stober method and HMSNs with an entire hollow core was induced by immersing cetyltrimethylammonium bromide (CTAB) in hot water. Initial MSNs were added in boiling distilled water to synthesize hollow structure, to enhance pore size, and also to remove CTAB template. HMSNs prepared in our current study has exhibited high surface area (886.84 m2/g), pore volume (0.79 cm3/g), and uniform pore size (3.18 nm), which also enabled the greater encapsulation of the model BSC II drug Carvedilol (CAR) inside the HMSNs. This technique also helped in achieving a high drug loading of (40.22 ± 0.73)%. Add to all this, in vitro studies conducted in the present work showed that compared with pure CAR and CAR loaded MSNs (CAR-MSNs) synthesized by Stober method, the drug-loaded HMSNs (CAR-HMSNs) exhibit sustained drug release performance. The high drug loading and sustained release can be attributed to the hollow porous structure of the HMSNs. Finally, a pharmacokinetic analysis in rats indicated a significant increase in bioavailability of carvedilol HMSNs in vivo compared to the pure carvedilol and carvedilol loaded MSNs. This study, therefore, offered a new, simple, and quick method to develop HMSNs with the ability to support higher loading and controlled release behavior in vitro and enhanced absorption of poorly-aqueous soluble drugs in-vivo.

Integrating the drug, disulfiram into the vitamin E-TPGS-modified PEGylated nanostructured lipid carriers to synergize its repurposing for anti-cancer therapy of solid tumors.

The 'repurposed drug,' disulfiram (DSF), is an inexpensive FDA-approved anti-alcoholism drug with multi-target anti-cancer effect. However, the use of DSF in clinical settings remains limited due to its high instability in blood. In the present study, we created nanostructured lipid carriers (NLC) encapsulated DSF modified with d-α-tocopheryl polyethylene glycol 1000 succinate (vitamin E-TPGS). A spherical shape, superior drug encapsulation (80.7%), and decreased crystallinity of DSF were confirmed with results obtained from TEM, XRD, and DSC analysis. Addition of TPGS considerably improved the physicochemical stability profile of NLC-encapsulated DSF under the different conditions tested here. Furthermore, TPGS-DSF-NLCs outperformed unmodified DSF-NLCs and the free DSF solution by having significantly higher cytotoxicity, lower IC50 value (4T1: 263.2 nM and MCF-7: 279.9 nM), and an enhanced cellular uptake in MCF7 and 4T1 cell lines. In vivo anti-tumor analysis in 4T1 murine xenograft model mice revealed a significant (p-value < 0.05) decrease in tumor volume and higher tumor growth inhibition rate (48.24%) with TPGS-DSF-NLC treatment as compared to both the free DSF solution (8.49%) and DSF-NLC formulations (29.2%). Histopathology analysis of tumor tissues further confirmed a noticeably higher anti-tumor activity of TPGS-DSF-NLC through augmented cell necrosis in solid tumors. Hence, the present study established that addition of TPGS can synergize the anti-cancer activity of NLC-encapsulated DSF formulations, and thus, offer a promising anti-cancer delivery system for DSF.

Comparative study on stabilizing ability of food protein, non-ionic surfactant and anionic surfactant on BCS type II drug carvedilol loaded nanosuspension: Physicochemical and pharmacokinetic investigation.

Carvedilol (CAR) in its pure state has low aqueous solubility and extremely poor bioavailability which largely limit its clinical application. The aim of the study is to improve the dissolution rate and the bioavailability of CAR via preparing nanosuspensions with different stabilizers. Antisolvent precipitation-ultrasonication technique was used here. Attempts have been made to use food protein- Whey protein isolate (WPI) as a stabilizer in CAR loaded nanosuspension and also to compare its stabilizing potential with conventional nanosuspension stabilizers such as non-ionic linear copolymer-poloxamer 188 (PLX188) and anionic surfactant-sodium dodecyl sulfate (SDS). Optimized nanosuspensions showed narrow size distribution with particle size ranging from 275 to 640nm. Amorphous state of CAR nanocrystals which also improved the solubility by 16-, 25-, 55-fold accordingly was confirmed by powder X-ray diffraction (PXRD) and differential scanning calorimetry (DSC). From scanning electron microscopy (SEM), flaky shape of PLX188 and SDS nanosuspensions could be revealed but WPI nanosuspension was sphere-shaped. Up to 70% dissolution of loaded drug was observed within 15min in phosphate buffer (pH6.8). A pharmacokinetic study in rats indicated that both Cmax and AUC0-36 values of nanosuspensions were estimated to be 2-fold higher than those of reference, suggesting a significant increase in CAR bioavailability.