The COVID-19 pandemic has left scientists and clinicians no choice but a race to find solutions to save lives while controlling the rapid spreading. Messenger RNA (mRNA)-based vaccines have become ...the front-runners because of their safety profiles, precise and reproducible immune response with more cost-effective and faster production than other types of vaccines. However, the physicochemical properties of naked mRNA necessitate innovative delivery technologies to ferry these ‘messengers’ to ribosomes inside cells by crossing various barriers and subsequently induce an immune response. Intracellular delivery followed by endosomal escape represents the key strategies for cytoplasmic delivery of mRNA vaccines to the target. This Perspective provides insights into how state-of-the-art nanotechnology helps break the delivery barriers and advance the development of mRNA vaccines. The challenges remaining and future perspectives are outlined.
This study aimed to develop redox-sensitive and CD44-targeted liposomes to improve chemotherapy of osteosarcoma. Cationic liposomes were prepared and stabilized with a novel detachable polyethylene ...glycol (PEG2000) conjugated with cholesterol through a bio-reducible disulfide linker (Chol-SS-mPEG). Hyaluronic acid (HA, MW 20–40kDa), a ligand to CD44, was non-covalently coated on the cationic liposomes. Doxorubicin (DOX) was actively loaded in the liposomes as a model drug. The roles of HA and Chol-SS-mPEG on intracellular drug delivery efficiency, and antitumor efficacy were studied. The structure of Chol-SS-mPEG was confirmed with Fourier-transform infrared and nuclear magnetic resonance (1H NMR). The liposomes, Chol-SS-mPEG/HA-L had a mean diameter of 165nm, zeta potential −28.9mV, and destabilized in reducing or acidic (pH5–6) conditions. In vitro release of DOX was well-controlled at physiological conditions, but a burst release of 60% was observed in the presence of 10mM glutathione (GSH), in contrast to non-redox sensitive liposomes (Chol-mPEG/HA-L and Chol-mPEG-L). MTT cell viability assay showed that the dual-functional Chol-SS-mPEG/HA-L with a drug loading of 15.0% (w/w) had significantly higher cytotoxicity to MG63 osteosarcoma cells compared with non-reduction sensitive or non-HA coated liposomes (p<0.01), consistent with the cellular uptake and intracellular trafficking studies using confocal microscopy and flow cytometry. Furthermore, the HA-coated GSH-responsive liposomes preferentially internalized to MG63 over human liver cells LO2. In rats, liposomes stabilized with either Chol-SS-mPEG or Chol-mPEG, with or without HA, increased the half-life of DOX by >10-fold. In a MG63 xenograft mouse model, Chol-SS-mPEG/HA-L showed the most effective tumor suppression with minimal uptake by the liver compared with other liposomes. All animals treated with liposomal formulations survived, in contrast to those free-DOX treated. In conclusion, the easily prepared Chol-SS-mPEG/HA-L was demonstrated as an excellent CD44-mediated intracellular delivery system capable of long-circulation and GSH-triggered cytoplasmic drug release. Further translational and multidisciplinary research is required to make it real clinical benefits to cancer patients.
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Purpose
PEGylated pH-sensitive liposomes (PSL) dual-loaded with gemcitabine and curcumin were investigated for the potential application in gemcitabine-resistant pancreatic ductal adenocarcinoma ...(PDAC) treatment. Curcumin was employed as an inhibitor of the efflux transporter, multidrug resistance protein 5 (MRP5) in PDAC cells.
Methods
Liposomes were prepared with gemcitabine in the core and curcumin in the bilayers. The effects of curcumin on pH-sensitivity and ‘endosome escape’ of PSL with different PEGylation were investigated using a calcein self-quench assay. The effects of curcumin on intracellular gemcitabine concentrations, and cytotoxicity to a MIA PaCa-2 PDAC cell line was evaluated. The pharmacokinetics were investigated in rats following intravenous injection.
Results
The addition of curcumin to the PSL bilayers (0.2-1 mol%)slightly decreased the pH-sensitivity of PSL, but to a less extent than PEGylation (0–5 mol%). Co-treatment with curcumin increased gemcitabine cellular accumulation in a concentration-dependent manner, and resulted in synergistic cytotoxicity towards MIA PaCa-2cells.Both these effects were augmented by the use of PSL, particularly when the two drugs were co-loaded in PSL. In rats, the dual-drug loaded PSL produced significantly reduced (
p
< 0.05) plasma clearance (CL) and volume of distribution (V
d
) for both drugs, alongside 3 to 4-fold increases in the area-under-the-concentration-time curves compared to the free drugs. Additionally, curcumin slightly increase the plasma concentrations of gemcitabine possibly also via the MRP5 inhibition effect.
Conclusion
Co-delivery of curcumin with gemcitabine using PSL not only increased the intracellular gemcitabine concentration thus cytotoxicity to MIA PaCa-2 cells but also significantly improved the pharmacokinetic profiles for both drugs.
Graphical Abstract
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.
Graphical abstract
Safe and effective delivery of therapeutic drugs to the brain is important for successful therapy of Alzheimer's disease (AD).
To develop Huperzine A (HupA)-loaded, mucoadhesive and targeted ...polylactide-co-glycoside (PLGA) nanoparticles (NPs) with surface modification by lactoferrin (Lf)-conjugated N-trimethylated chitosan (TMC) (HupA Lf-TMC NPs) for efficient intranasal delivery of HupA to the brain for AD treatment.
HupA Lf-TMC NPs were prepared using the emulsion-solvent evaporation method and optimized using the Box-Behnken design. The particle size, zeta potential, drug entrapment efficiency, adhesion and in vitro release behavior were investigated. The cellular uptake was investigated by fluorescence microscopy and flow cytometry. MTT assay was used to evaluate the cytotoxicity of the NPs. In vivo imaging system was used to investigate brain targeting effect of NPs after intranasal administration. The biodistribution of Hup-A NPs after intranasal administration was determined by liquid chromatography-tandem mass spectrometry.
Optimized HupA Lf-TMC NPs had a particle size of 153.2±13.7 nm, polydispersity index of 0.229±0.078, zeta potential of +35.6±5.2 mV, drug entrapment efficiency of 73.8%±5.7%, and sustained release in vitro over a 48 h period. Adsorption of mucin onto Lf-TMC NPs was 86.9%±1.8%, which was significantly higher than that onto PLGA NPs (32.1%±2.5%). HupA Lf-TMC NPs showed lower toxicity in the 16HBE cell line compared with HupA solution. Qualitative and quantitative cellular uptake experiments indicated that accumulation of Lf-TMC NPs was higher than nontargeted analogs in 16HBE and SH-SY5Y cells. In vivo imaging results showed that Lf-TMC NPs exhibited a higher fluorescence intensity in the brain and a longer residence time than nontargeted NPs. After intranasal administration, Lf-TMC NPs facilitated the distribution of HupA in the brain, and the values of the drug targeting index in the mouse olfactory bulb, cerebrum (with hippocampus removal), cerebellum, and hippocampus were about 2.0, 1.6, 1.9, and 1.9, respectively.
Lf-TMC NPs have good sustained-release effect, adhesion and targeting ability, and have a broad application prospect as a nasal drug delivery carrier.
Chemoresistance poses a major challenge in cancer treatment. This study aims to investigate whether intracellular drug delivery using hyaluronic acid (HA) functionalised pH-sensitive liposomes ...(HA-pSL) can circumvent gemcitabine resistance in pancreatic cancer (PC). HA-pSL were obtained by covalently conjugating HA with preformed pSL. A resistant PC cell line Gr2000 was developed by exposing MIA PaCa-2 cells to gemcitabine, and characterised for their expression of CD44, a receptor for HA, and drug transporters. Cellular uptake and intracellular trafficking of liposomes were determined by confocal microscopy and HPLC analysis of intracellular drug content. Following a pharmacokinetic study in rats, anti-tumour efficacy was compared between MIA PaCa-2 and Gr2000 xenograft mouse models. HA-pSL with an HA density of 179 μg/μmol had a larger size (152.3 vs 136.3 nm), and higher zeta potential (−46.8 vs −10.5 mV) than pSL. The sensitivity of Gr2000 to gemcitabine reduced 444 times compared to its parental cell line, despite no change to the total drug influx, as drug influx- and efflux-transporters in Gr2000 cells were simultaneously up-regulated. Both cell lines had high expression of CD44. HA facilitated cell uptake without compromising the endosome-escape ability of pSL as evidenced by confocal images and co-localization analysis of the dual-fluorescence labelled liposomes and Lysotracker. HA-pSL significantly outperformed pSL, and increased cellular drug influx by 3.6 times in MIA PaCa-2 cells, and 4.6 times in Gr2000 cells. Both liposomes improved the pharmacokinetic profile of free drug. HA-pSL treatment was superior to pSL, and resulted in 6.4 times smaller tumours (weight) in the MIA PaCa-2 xenograft models, and 3.1 smaller in the Gr2000 models compared with the free drug. Taken together, this study highlighted the use of intracellular delivery strategies (HA-CD44 interaction and endosome escape) to overcome gemcitabine resistance, however, the overall improvement was marginal and tumours still existed. Further improvement in delivery efficiency of HA-pSL to target tumours and additional manipulation of the cellular metabolism of gemcitabine are needed to tackle chemoresistance.
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•Gr2000 were MIA PaCa-2 pancreatic cancer cells acquired gemcitabine-resistance.•Gr2000 retained the high expression of CD44 and had altered drug transporters.•HA facilitated the cellular uptake of pSL without compromising endosome escape.•HA-pSL were 6.4 times more potent than free gemcitabine to MIA PaCa-2 tumours.•HA-pSL were only 3.1 times more potent than free gemcitabine to Gr2000 tumours.
Poisoning is a significant cause of injury-related death worldwide. Dialysis is usually ineffective in removing the toxin once it has been absorbed because of drug protein binding and high volumes of ...distribution. In this work, we explore whether the addition of liposomes to peritoneal dialysate could extract protein bound amitriptyline. Liposomes were prepared using the thin film hydration method. In the in vitro experiment, 3 mL of 20% albumin with a concentration of 6000 nmol/L amitriptyline in a proprietary dialysis cartridge was dialysed against 125 mL of phosphate-buffered saline with and without 80 mg 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) liposomes. In the in vivo arm, peritoneal dialysis was undertaken in 6 rats with pH gradient liposome augmented dialysate after intravenous amitriptyline injection. Peritoneal blood flow was estimated by CO2 extraction. Total amitriptyline extracted was compared to freely dissolved (non-protein bound) and total amitriptyline perfusing the membrane during the peritoneal dwell. Mean liposome size for DOPG and acidic centre pH gradient liposomes was 119 nm and 430 nm, respectively. In the in vitro experiment, more amitriptyline was extracted into the liposome containing dialysate than the control dialysate (40 +/− 2 nmol/L vs. 27 +/− 1 nmol/L). In the in vivo experiment, the total amitriptyline in dialysate was 5240 +/− 2750 nmol. Mean total free amitriptyline perfusing the peritoneal membrane was 93 +/− 46 nmol. Mean total blood amitriptyline perfusing the peritoneal membrane was 23,920 +/− 6920 nmol. Two of the six animals were excluded due to overestimation of peritoneal blood flow. This exploratory work suggests the addition of liposome nanoparticles to peritoneal dialysate extracted protein bound amitriptyline from blood.
This study aimed to develop an efficient step-by-step osteosarcoma (OS)-targeting liposome system functionalized with a redox-cleavable, bone- and cluster of differentiation 44 (CD44)-dual-targeting ...polymer. Furthermore, the effect of coadministration of a tumor-penetrating peptide, internalizing RGD (iRGD), was investigated. First, a bone-targeting moiety, alendronate (ALN), was conjugated with hyaluronic acid (HA), a ligand for CD44. This ALN–HA conjugate was coupled with DSPE–PEG2000–COOH through a bioreducible disulfide linker (−SS−) to obtain a functionalized lipid, ALN–HA–SS–L, to be postinserted into preformed liposomes loaded with doxorubicin (DOX). The roles of ALN, HA, and the redox sensitivity of the ALN–HA–SS–L liposomes (ALN–HA–SS–L–L) in the anti-OS effect were critically evaluated against various reference liposomal formulations (with only ALN, HA, or redox sensitivity). ALN–HA–SS–L–L displayed a zeta potential of −26.07 ± 0.32 mV and selectively disassembled in the presence of a reducing agent, 10 mM glutathione, which can be found in cancer cells. Compared to various reference liposomes, ALN–HA–SS–L–L/DOX had significantly higher cytotoxicity to human OS MG-63 cells alongside high and rapid cellular uptake. In the orthotopic OS nude mouse models, ALN–HA–SS–L–L/DOX showed remarkable tumor growth suppression and prolonged survival time. This result was further improved by the coadministration of iRGD. The antitumor effects of various liposomes were ranked in the same order as the degree of tumor biodistribution shown by in vivo/ex vivo imaging: ALN–HA–SS–L–L coadministered with iRGD > ALN–HA–SS–L–L > HA–SS–L–L > HA–L–L > PEG–L> free drug. ALN–HA–SS–L–L/DOX also reduced the cardiotoxicity of DOX and lung metastases. Overall, this study demonstrated that ALN–HA–SS–L–L/DOX, equipped with bone- and CD44-dual-targeting abilities and redox sensitivity, could be a promising OS-targeted therapy. The efficacy could also be augmented by coadministration of iRGD.
An estrogen (ES)-functionalized cationic liposomal system was developed and exploited for targeted delivery to osteosarcoma. Natural biocompatible chotooligosaccharides (COS, MW2-5 KDa) were ...covalently tethered to the liposomal surface through a disulfate bond (-SS-) to confer reduction-responsive COS detachment, whereas estrogen was grafted via polyethylene glycol (PEG 2 K) chain to achieve estrogen receptor-targeting. The liposomal carriers were prepared by the ethanol injection method and fluorescent anticancer drug doxorubicin (DOX) was loaded with ammonium sulfate gradient. The physicochemical properties, reduction-sensitivity, and the roles of estrogen on cellular uptake and tumor-targeting were studied. The Chol-SS-COS/ES/DOX liposomes were spherical with an average size about 110 nm, and high encapsulation efficiency. The liposomes were stable in physiological condition but rapidly release the payload in response to tumoral intracellular glutathione (20 mM). MTT cytotoxicity assay confirmed that Chol-SS-COS/ES/DOX liposomes exhibited higher cytotoxicity to MG63 osteosarcoma cells than to liver cells (LO2). Flow cytometry (FCM) and confocal laser scanning microscopy revealed that cellular uptake of Chol-SS-COS/ES/DOX liposomes by MG63, than the free DOX or Chol-SS-COS/DOX. Ex vivo fluorescence distribution study showed that the multifunctional liposomes selectively accumulated in the MG63 xenografts versus the organs. Chol-SS-COS/ES/DOX liposomes strongly inhibited the tumor growth and enhanced the animal survival rate. Overall, the COS grafted estrogen-functionalized cationic liposomes, fortified with glutathione-responsiveness, showed great potential for specific intracellular drug delivery to estrogen receptor-expressing tumors such as osteosarcoma.
Efficient delivery of brain-targeted drugs is highly important for successful therapy in Parkinson's disease (PD). This study was designed to formulate borneol and lactoferrin co-modified ...nanoparticles (Lf-BNPs) encapsulated dopamine as a novel drug delivery system to achieve maximum therapeutic efficacy and reduce side effects for PD. Dopamine Lf-BNPs were prepared using the double emulsion solvent evaporation method and evaluated for physicochemical and pharmaceutical properties. In vitro cytotoxicity studies indicated that treatment with dopamine Lf-BNPs has relatively low cytotoxicity in SH-SY5Y and 16HBE cells. Qualitative and quantitative cellular uptake experiments indicated that Lf modification of NPs increased cellular uptake of SH-SY5Y cells and 16HBE cells, and borneol modification can promote the cellular uptake of 16HBE. In vivo pharmacokinetic studies indicated that AUC
0-12 h
in the rat brain for dopamine Lf-BNPs was significantly higher (p < .05) than that of dopamine nanoparticles. Intranasal administration of dopamine Lf-BNPs effectively alleviated the 6-hydroxydopamine-induced striatum lesion in rats as indicated by the contralateral rotation behavior test and results for striatal monoamine neurotransmitter content detection. Taken together, intranasal administration of dopamine Lf-BNPs may be an effective drug delivery system for Parkinson's disease.
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