Efficient Intracellular Drug Release Research Articles

A comprehensive review of engineered exosomes from the preparation strategy to therapeutic applications.

Exosomes exhibit high bioavailability, biological stability, targeted specificity, low toxicity, and low immunogenicity in shuttling various bioactive molecules such as proteins, lipids, RNA, and DNA. Natural exosomes, however, have limited production, targeting abilities, and therapeutic efficacy in clinical trials. On the other hand, engineered exosomes have demonstrated long-term circulation, high stability, targeted delivery, and efficient intracellular drug release, garnering significant attention. The engineered exosomes bring new insights into developing next-generation drug delivery systems and show enormous potential in therapeutic applications, such as tumor therapies, diabetes management, cardiovascular disease, and tissue regeneration and repair. In this review, we provide an overview of recent advancements associated with engineered exosomes by focusing on the state-of-the-art strategies for cell engineering and exosome engineering. Exosome isolation methods, including traditional and emerging approaches, are systematically compared along with advancements in characterization methods. Current challenges and future opportunities are further discussed in terms of the preparation and application of engineered exosomes.

Construction of Enzyme-Responsive Micelles Based on Theranostic Zwitterionic Conjugated Bottlebrush Copolymers with Brush-on-Brush Architecture for Cell Imaging and Anticancer Drug Delivery.

Bottlebrush copolymers with different chemical structures and compositions as well as diverse architectures represent an important kind of material for various applications, such as biomedical devices. To our knowledge, zwitterionic conjugated bottlebrush copolymers integrating fluorescence imaging and tumor microenvironment-specific responsiveness for efficient intracellular drug release have been rarely reported, likely because of the lack of an efficient synthetic approach. For this purpose, in this study, we reported the successful preparation of well-defined theranostic zwitterionic bottlebrush copolymers with unique brush-on-brush architecture. Specifically, the bottlebrush copolymers were composed of a fluorescent backbone of polyfluorene derivate (PFONPN) possessing the fluorescence resonance energy transfer with doxorubicin (DOX), primary brushes of poly(2-hydroxyethyl methacrylate) (PHEMA), and secondary graft brushes of an enzyme-degradable polytyrosine (PTyr) block as well as a zwitterionic poly(oligo (ethylene glycol) monomethyl ether methacrylate-co-sulfobetaine methacrylate) (P(OEGMA-co-SBMA)) chain with super hydrophilicity and highly antifouling ability via elegant integration of Suzuki coupling, NCA ROP and ATRP techniques. Notably, the resulting bottlebrush copolymer, PFONPN9-g-(PHEMA15-g-(PTyr16-b-P(OEGMA6-co-SBMA6)2)) (P2) with a lower MW ratio of the hydrophobic side chains of PTyr and hydrophilic side chains of P(OEGMA-co-SBMA) could self-assemble into stabilized unimolecular micelles in an aqueous phase. The resulting unimolecular micelles showed a fluorescence quantum yield of 3.9% that is mainly affected by the pendant phenol groups of PTyr side chains and a drug-loading content (DLC) of approximately 15.4% and entrapment efficiency (EE) of 90.6% for DOX, higher than the other micelle analogs, because of the efficient supramolecular interactions of π–π stacking between the PTyr blocks and drug molecules, as well as the moderate hydrophilic chain length. The fluorescence of the PFONPN backbone enables fluorescence resonance energy transfer (FRET) with DOX and visualization of intracellular trafficking of the theranostic micelles. Most importantly, the drug-loaded micelles showed accelerated drug release in the presence of proteinase K because of the enzyme-triggered degradation of PTyr blocks and subsequent deshielding of P(OEGMA-co-SBMA) corona for micelle destruction. Taken together, we developed an efficient approach for the synthesis of enzyme-responsive theranostic zwitterionic conjugated bottlebrush copolymers with a brush-on-brush architecture, and the resulting theranostic micelles with high DLC and tumor microenvironment-specific responsiveness represent a novel nanoplatform for simultaneous cell image and drug delivery.

An Integrated Tumor Microenvironment Responsive Polymeric Micelle for Smart Drug Delivery and Effective Drug Release.

Tumor microenvironment (TME) responsive polymeric micelles are promising carriers for drug delivery. In order to meet the needs of various applications, multifarious TME-responsive switches are used to construct smart polymeric micelles, which causes the complexity and corpulence of the polymeric micelle system and increases the difficulty of preparation. In this study, we designed and synthesized an ingenious TME-responsive switch through grafting disulfide bond-modified piperidinepropionic acid (CPA) on copolymer poly(ethylene glycol)-b-poly(aspartate)(PEG-b-PAsp) and built a novel pH/reduction-responsive PEG-b-PAsp-g-CPA polymeric micelle delivery system. The CPA-pendants can reverse the surface charge of the polymeric micelle from negative to positive at pH 6.5 because of the protonation of piperidine groups, thereby enhancing the internalization of cell. Subsequently, more piperidine groups are protonated at pH 5.0 which will increase the hydrophilicity of polymeric micelles and cause the hydrophobic core to swell, thus making the disulfide bonds packed in the core to be more easily broken by GSH. With the synergistic effect of the pH-triggered protonation of piperidine groups and reduction triggered break of disulfide bonds, the polymeric micelles will disintegrate and achieve efficient intracellular drug release. The TME-responsive polymeric micelles exhibited good biological safety, enhanced internalization, and rapid intracellular doxorubicin (DOX) release in vitro. Moreover, the PEG-b-PAsp-g-CPA/DOX polymeric micelles showed excellent antitumor efficacy and low systemic toxicity in lung tumor-bearing BALB/C mice. These results indicated that the novel integrated TME-responsive switch CPA helps the PEG-b-PAsp-g-CPA polymeric micelles to obtain excellent TME-responsiveness and antitumor drug delivery capabilities, while it also makes the preparation of TME-responsive polymeric micelles simpler and more convenient. This work provides a new idea for the architecture of TME-responsive polymeric micelles.

Hybrid micelles enhance tumour therapy by remodelling biodistribution and improving intracellular drug release.

PEGylated micelles have been widely used for tumour therapy. Although PEGylation can prolong the blood circulation time, there is only less than 5% of administered micelles that can be transported to tumour sites and over 95% are cleared by the reticuloendothelial system (RES). Besides, the limited intracellular drug release also restricts their efficacy. To improve the therapeutic efficacy of PEGylated micelles, a safe, simple and efficient hybrid micellar system, composed of poly(aminoethyl ethylene phosphate)-poly(L-lactic acid) (PAEEP-PLLA) and poly(ethylene glycol)-poly(L-lactic acid) (PEG-PLLA), was developed. The hybrid micelles significantly prolonged the blood circulation time by decreasing the plasma protein adsorption and reducing the clearance by the RES. The deposition of the hybrid micelles in the liver and spleen was reduced, and the tumour accumulation was greatly improved. In addition, the intracellular drug release of the hybrid micelles was obviously increased due to the easy degradation of PAEEP in the endo/lysosomes. The tumour growth inhibition efficiency of the hybrid micelles was much higher than that of the PEG-PLLA micelles (84.5% vs. 44.5%). Furthermore, the hybrid micelles exhibited low hemolysis and reduced deposition in normal organs, which revealed their excellent bio-safety. Therefore, we established a promising hybrid micelle system for efficient anti-tumour therapy.

Polycaprolactone-Based Mimetic Antimicrobial Peptide Copolymers Vesicles as an Effective Drug-Carrier for Cancer Therapy.

A novel series of amphiphilic mimicking antimicrobial peptide copolymers PCL16-b-Kn can assemble in water to form uniform vesicles. Transmission electron microscopy was used to observe the vesicular structure of the nanoparticles, and dynamic light scattering revealed their uniform size and narrow dispersion. Critical vesiculation concentrations were also tested, revealing that these vesicles can exist at low concentrations. Furthermore, in vitro and intracellular drug release of doxorubicin(DOX)-vesicles were conducted. These vesicles could encapsulate DOX and achieve efficient intracellular drug release. Overall, these copolymer vesicles exhibit potential application value as multifunctional drug-carrier systems with antibacterial capability in cancer therapy.

Self-Assembled Indomethacin Dimer Nanoparticles Loaded with Doxorubicin for Combination Therapy in Resistant Breast Cancer.

An ortho-ester-linked indomethacin (IND) dimer-based nanodrug delivery system was prepared to improve the therapeutic effect of doxorubicin (DOX) by reversing the multidrug resistance. The synthesized dimer (IND-OE) could form stable nanoparticles (IND-OE/DOX) loaded with DOX via the single-emulsion method. Compare to insensitive nanoparticles (IND-C12/DOX), IND-OE/DOX showed a rapid degradation behavior and accelerated drug release at mildly acidic environments. In vitro cell experiments verified that IND-OE nanoparticles could increase DOX concentration due to the efficient intracellular drug release by the degradation of the ortho ester as well as reduced DOX efflux by IND-mediated P-gp downregulation. In vivo studies further demonstrated that IND-OE/DOX displayed the maximized synergetic antitumor efficacy than free DOX or IND-C12/DOX, and the tumor inhibition rates versus saline were 46.78% (free DOX), 60.23% (IND-C12/DOX), and 80.62% (IND-OE/DOX). Overall, this strategy of combination with chemosensitizers and ortho ester linkage has great potential to serve as an amplifying chemotherapy platform against various drug-resistant tumors.

Conjugates of Cryptophycin and RGD or isoDGR Peptidomimetics for Targeted Drug Delivery.

RGD‐cryptophycin and isoDGR‐cryptophycin conjugates were synthetized by combining peptidomimetic integrin ligands and cryptophycin, a highly potent tubulin‐binding antimitotic agent across lysosomally cleavable Val‐Ala or uncleavable linkers. The conjugates were able to effectively inhibit binding of biotinylated vitronectin to integrin αvβ3, showing a binding affinity in the same range as that of the free ligands. The antiproliferative activity of the novel conjugates was evaluated on human melanoma cells M21 and M21‐L with different expression levels of integrin αvβ3, showing nanomolar potency of all four compounds against both cell lines. Conjugates containing uncleavable linker show reduced activity compared to the corresponding cleavable conjugates, indicating efficient intracellular drug release in the case of cryptophycin‐based SMDCs. However, no significant correlation between the in vitro biological activity of the conjugates and the integrin αvβ3 expression level was observed, which is presumably due to a non‐integrin‐mediated uptake. This reveals the complexity of effective and selective αvβ3 integrin‐mediated drug delivery.

TPGS-grafted and acid-responsive soy protein nanogels for efficient intracellular drug release, accumulation, penetration in 3D tumor spheroids of drug-resistant cancer cells

The frequent occurrence of multidrug resistance (MDR) in solid tumors is the major obstacle for nano-drug delivery systems (nDDS) to realize the successful cancer chemotherapy. Herein, we had prepared pH-responsive nanogels via cross-linking TPGS-grafted soy protein with an acid-labile ortho ester cross-linker (OEAM) to realize the efficient intracellular drugs release and accumulation, and subsequently enhance therapeutic effect in MDR tumor cells. These nanogels displayed a uniform size (~200 nm) and morphology, and the introduction of ortho ester bonds endowed nanogels stability in neutral environment and acid-degradability in acidic conditions. Cisplatin (CDDP) was successfully loaded into nanogels, which exhibited an accelerated drug release at low pH. The modification of TPGS efficiently improved cellular internalization and drug accumulation in A549/DDP cells by inhibiting the function of drug efflux pumps (MRP2 and ATP7A/7B), leading to higher cytotoxicity and apoptosis. Moreover, TPGS-grafted nanogels also showed better drug accumulation and penetration in tumor-like spheroids, and then remarkably inhibited tumor growth owing to the rapid drug release in acidic organelles. As a result, the TPGS-grafted and pH-sensitive soy protein nanogels have a great potential as a drugs carrier for the efficient cancer treatment.

PH-sensitive nanogels with ortho ester linkages prepared via thiol-ene click chemistry for efficient intracellular drug release

pH-sensitive nanogels with ortho ester linkages were conveniently prepared through reaction of thiol-ene click chemistry. Through adjusting feed reactant ratios and concentrations of ortho ester diacrylamide (OEAM), pentaerythritol tetra(3-mercaptopropionate) (PT), and methoxyl poly(ethyleneglycol) acrylate (mPEGA), the size of the nanogels could be controlled at 100–200nm with relatively narrow size distributions. The nanogels with size of 149.1±17.7nm (designed as NG) were verified by proton nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FT-IR), dynamic laser scattering (DLS) and transmission electron microscopy (TEM). Doxorubicin (DOX) was loaded into NG with high drug loading efficiency up to 73.7%. In vitro drug release studies showed that up to 75.9% DOX from NG was released in 24h at pH 5.0 because of hydrolysis of ortho ester. Cellular uptake studies confirmed that DOX-loaded NG (NG/DOX) could be readily internalized by two-dimensional cells, resulting in efficient antitumor efficiency of cancer cells. Three-dimensional (3D) multicellular tumor spheroids (MCTS) as in vitro tumor model was used to further evaluate the antitumor effect of NG/DOX. The results demonstrated that NG/DOX showed a significantly enhanced penetration and growth inhibition in 3D multicellular tumor spheroids (MCTS), compared to free DOX.

Suppression of human arthritis synovial fibroblasts inflammation using dexamethasone-carbon nanotubes via increasing caveolin-dependent endocytosis and recovering mitochondrial membrane potential.

Dexamethasone (DEX), a non-particulate glucocorticoid (GC) to inhibit anti-inflammatory response, has been widely used for the treatment of various diseases such as arthritis, cancer, asthma, chronic obstructive pulmonary disease, cerebral edema, and multiple sclerosis. However, prolonged and/or high-dose GC therapy can cause various serious adverse effects (adrenal insufficiency, hyperglycemia, Cushing’s syndrome, osteoporosis, Charcot arthropathy, etc). In this study, developed DEX-carbon nanotube (CNT) conjugates improved intracellular drug delivery via increased caveolin-dependent endocytosis and ultimately suppressed the expression of major pro-inflammatory cytokines in tumor necrosis factor-α (TNF-α)-stimulated human fibroblast-like synoviocytes (FLS) at low drug concentrations. Specifically, DEX on polyethylene-glycol (PEG)-coated CNTs induced caveolin uptake, recovered mitochondrial disruption, and inhibited reactive oxygen species production by targeting mitochondria that was released from the early endosome in TNF-α-stimulated FLS. The obtained results clearly demonstrated that DEX-PEG-coated CNTs significantly inhibited the inflammation by FLS in rheumatoid arthritis (RA) by achieving greater drug uptake and efficient intracellular drug release from the endosome, thus suggesting a mechanism of effective low-dose GC therapy to treat inflammatory diseases, including RA and osteoarthritis.

Curcumin-loaded redox response of self-assembled micelles for enhanced antitumor and anti-inflammation efficacy.

At present, it has become evident that inflammation plays a critical role in tumor growth; meanwhile, chemotherapeutic agents using nanocarriers have been suggested as a promising strategy in cancer treatment. In this study, novel redox-responsive micelles were prepared from monomethoxy-poly(ethylene glycol)-chitosan-S-S-hexadecyl (C16-SS-CS-mPEG). These micelles were able to carry and deliver drugs into tumor cells. To serve as a control, monomethoxy-poly(ethylene glycol)-chitosan-C-C-hexadecyl (C16-CC-CS-mPEG) was developed in a similar fashion to that used to yield C16-CC-CS-mPEG without a redox-responsive disulfide bond. The cellular uptake mechanisms of both micelles were determined. The efficient intracellular drug release from micelles in MCF-7 cells was further confirmed. Results indicated that curcumin (Cur) could rapidly form C16-SS-CS-mPEG@ Cur micelles when exposed to reducing agents and efficaciously enhance intracellular accumulation. The cytotoxicity assay demonstrated that C16-SS-CS-mPEG@Cur exhibited satisfactory cytotoxicity against MCF-7 cells. Anti-inflammation assay results indicated that C16-SS-CS-mPEG@Cur treatment significantly downregulated tumor necrosis factor (TNF-α) expression and showed good anti-inflammatory effects in tumor microenvironment. Most importantly, antitumor effects in vivo showed satisfactory therapeutic effects with C16-SS-CS-mPEG@Cur. Hence, C16-SS-CS-mPEG@Cur micelles can be useful in tumor therapy.

Biological evaluation of redox-sensitive micelles based on hyaluronic acid-deoxycholic acid conjugates for tumor-specific delivery of paclitaxel

Tumor-targeted drug delivery and microenvironment-responsive drug release are attractive strategies in cancer treatment. Our previous study demonstrated that redox-sensitive micelles based on hyaluronic acid-deoxycholic acid (HA-ss-DOCA) conjugates exhibited excellent drug-loading capacities (34.1%) for paclitaxel (PTX) and rapid drug release in response to reducing agent, glutathione. In the present study, the physicochemical and biological properties of PTX-loaded HA-ss-DOCA (PTX-HA-ss-DOCA) micelles were investigated further. The micelles have an average size of about 120nm and a zeta potential of about −36mV. Transmission electron microscopy and wide-angle X-ray diffraction analysis demonstrated redox-sensitive degradation of micelles in the presence of glutathione. Moreover, the encapsulated payload was effectively released from HA-ss-DOCA micelles into cytoplasm and then rapidly transported into nuclei. In vitro cytotoxicity and cell apoptosis assay further revealed that HA significantly improved the tumor-specific drug delivery of HA-ss-DOCA micelles via receptor-mediated endocytosis, while efficient intracellular drug release and transportation lead to marked inhibition of tumor cell growth, as compared to Taxol® and insensitive micelles. More importantly, PTX-HA-ss-DOCA micelles demonstrated superior in vivo antitumor activity compared with Taxol® and insensitive control, and decreased systemic toxicity. Herein we present data which provide valuable insight into the design and development of tumor-specific drug delivery systems.

Redox and pH-responsive poly (amidoamine) dendrimer–poly (ethylene glycol) conjugates with disulfide linkages for efficient intracellular drug release

Nanocarriers with low toxicity, high stability, long circulation in blood and triggered drug release at target sites are extremely needed for cancer therapy. In this study, different molar ratios of poly (ethylene glycol) (PEG) were conjugated to poly (amidoamine) dendrimer (PAMAM) by cleavable disulfide bonds, yielding redox and pH dual-responsive nanocarriers (PSSP). The redox sensitivity of the PSSP conjugates was confirmed by measuring the change in particle size and zeta potential under reductive environment by dynamic light scattering (DLS). Meanwhile, all conjugates showed slight hemolytic ratio and high stability in 10% FBS for 24h. Phagocytosis experiments indicated that the PSSP conjugates could effectively escape the engulfment of macrophage. Doxorubicin (DOX) was loaded into the hydrophobic core of the conjugates with loading content of 10%. In vitro release studies suggested that release rate as well as cumulative release amount of DOX from the PSSP conjugates were pronouncedly enhanced in reductive environment as compared with the non-cleavable counterparts. Besides, all conjugates exhibited excellent acid-triggered release performance. Significantly, PEGylation degree of the conjugates had critical impact on drug release. Cytotoxicity tests displayed that DOX-loaded PSSP conjugates were more potent in killing B16 and A549 tumor cells than the non-cleavable counterparts. Curiously, PSSP conjugate with the highest PEGylation degree showed the best antitumor effect despite the somewhat less cell uptake. Therefore, the dual-responsive PSSP conjugates seem promising candidates for efficient intracellular release of antitumor drugs.

Nano-Sized Albumin-Copolymer Micelles for Efficient Doxorubicin Delivery

We present the discovery of a nano-sized protein-derived micellar drug delivery system based on the polycationic albumin precursor protein cBSA-147. The anticancer drug doxorubicin (DOX) was efficiently encapsulated into nanosized micelles based on hydrophobic interactions with the polypeptide scaffold. These micelles revealed attractive stabilities in various physiological buffers and a wide pH range as well as very efficient uptake into A549 cells after 1 h incubation time only. In vitro cytotoxicity was five-times increased compared to free DOX also indicating efficient intracellular drug release. In addition, multiple functional groups are available for further chemical modifications. Based on the hydrophobic loading mechanism, various classical anti-cancer drugs, in principle, could be delivered even synergistically in a single micelle. Considering these aspects, this denatured albumin-based drug delivery system represents a highly attractive platform for nanomedicine approaches towards cancer therapy.

Glutathione-responsive nano-vehicles as a promising platform for targeted intracellular drug and gene delivery

The past couple of years have witnessed a tremendous progress in the development of glutathione-responsive nano-vehicles for targeted intracellular drug and gene delivery, as driven by the facts that (i) many therapeutics (e.g. anti-cancer drugs, photosensitizers, and anti-oxidants) and biotherapeutics (e.g. peptide and protein drugs, and siRNA) exert therapeutical effects only inside cells like the cytosol and cell nucleus, and (ii) several intracellular compartments such as cytosol, mitochondria, and cell nucleus contain a high concentration of glutathione (GSH) tripeptides (about 2–10 mM), which is 100 to 1000 times higher than that in the extracellular fluids and circulation (about 2–20 μM). Glutathione has been recognized as an ideal and ubiquitous internal stimulus for rapid destabilization of nano-carriers inside cells to accomplish efficient intracellular drug release. In this paper, we will review recent results on GSH-responsive nano-vehicles in particular micelles, nanoparticles, capsules, polymersomes, nanogels, dendritic and macromolecular drug conjugates, and nano-sized nucleic acid complexes for controlled delivery of anti-cancer drugs (e.g. doxorubicin and paclitaxel), photosensitizers, anti-oxidants, peptides, protein drugs, and nucleic acids (e.g. DNA, siRNA, and antisense oligodeoxynucleotide). The unique disulfide chemistry has enabled novel and versatile designs of multifunctional delivery systems addressing both intracellular and extracellular barriers. We are convinced that GSH-responsive nano-carrier systems have enormous potential in targeted cancer therapy.