Intracellular Delivery Of Doxorubicin Research Articles

Synthesis of silver nanoparticles for the dual delivery of doxorubicin and alendronate to cancer cells.

We present the synthesis of a silver nanoparticle (AgNP) based drug-delivery system that achieves the simultaneous intracellular delivery of doxorubicin (Dox) and alendronate (Ald) and improves the anticancer therapeutic indices of both drugs. Water, under microwave irradiation, was used as the sole reducing agent in the size-controlled, bisphosphonate-mediated synthesis of stabilized AgNPs. AgNPs were coated with the bisphosphonate Ald, which templated nanoparticle formation and served as a site for drug attachment. The unreacted primary ammonium group of Ald remained free and was subsequently functionalized with either Rhodamine B (RhB), through amide formation, or Dox, through imine formation. The RhB-conjugated NPs (RhB-Ald@AgNPs) were studied in HeLa cell culture. Experiments involving the selective inhibition of cell membrane receptors were monitored by confocal fluorescence microscopy and established that macropinocytosis and clathrin-mediated endocytosis were the main mechanisms of cellular uptake. The imine linker of the Dox-modified nanoparticles (Dox-Ald@AgNPs) was exploited for acid-mediated intracellular release of Dox. We found that Dox-Ald@AgNPs had significantly greater anti-cancer activity in vitro than either Ald or Dox alone. Ald@AgNPs can accommodate the attachment of other drugs as well as targeting agents and therefore constitute a general platform for drug delivery.

Reactive oxygen species (ROS) responsive PEG-PCL nanoparticles with pH-controlled negative-to-positive charge reversal for intracellular delivery of doxorubicin.

Nanocarriers have been extensively explored for cancer drug delivery with their ability to respond to cancer heterogeneity which is recently recognized as a critical doorway to a high therapeutic index. We proposed to develop a polycaprolactone bearing acid-labile β-carboxylic amide segments with charge reversal properties, which was coupled to mPEG with thioether as a linker. The linker could respond to overproduced reactive oxygen species (ROS) of cancer cells (ROS, e.g., perhaps more than one order of magnitude higher than healthy cells). This tailor-made surface charged nanoparticles (NPs) exhibited a capacity of reversing its surface charge from negative to positive at a tumor extracellular environment (pH ∼ 6.8) for enhancing cell internalization and an ability of response to the tumor ROS heterogeneity at the tumor intracellular environment to accelerate the release of drugs from NPs. The in vitro release studies showed that DOX release was greatly accelerated under the intracellular prevailing ROS (hydrogen peroxide (H2O2) simulating the oxidative stress). Cell uptake showed that the NPs could be more effectively internalized at pH 6.8 (simulating tumor extracellular conditions) than at pH 7.4. The MTT assay demonstrated that the DOX loaded NPs showed significant cytotoxicity to HepG2 cancer cells while no influence on the L02 normal cells. These ROS sensitive and surface charged NPs with superior cell internalization ability and rapid intracellular drug release provided a novel platform for tumor-targeting drug delivery.

Reductively degradable α-amino acid-based poly(ester amide)-graft-galactose copolymers: facile synthesis, self-assembly, and hepatoma-targeting doxorubicin delivery.

Novel reductively degradable α-amino acid-based poly(ester amide)-graft-galactose (SSPEA-Gal) copolymers were designed and developed to form smart nano-vehicles for active hepatoma-targeting doxorubicin (DOX) delivery. SSPEA-Gal copolymers were readily synthesized via solution polycondensation reaction of di-p-toluenesulfonic acid salts of bis-l-phenylalanine 2,2-thiodiethanol diester and bis-vinyl sulfone functionalized cysteine hexanediol diester with dinitrophenyl ester of adipic acid, followed by conjugating with thiol-functionalized galactose (Gal-SH) via the Michael addition reaction. SSPEA-Gal formed unimodal nanoparticles (PDI = 0.10 - 0.12) in water, in which average particle sizes decreased from 138 to 91 nm with increasing Gal contents from 31.6 wt% to 42.5 wt%. Notably, in vitro drug release studies showed that over 80% DOX was released from SSPEA-Gal nanoparticles within 12 h under an intracellular mimicking reductive conditions, while low DOX release (<20%) was observed for reduction-insensitive PEA-Gal nanoparticles under otherwise the same conditions and SSPEA-Gal nanoparticles under non-reductive conditions. Notably, SSPEA-Gal nanoparticles exhibited high specificity to asialoglycoprotein receptor (ASGP-R)-overexpressing HepG2 cells. MTT assays using HepG2 cells showed that DOX-loaded SSPEA-Gal had a low half maximal inhibitory concentration (IC50) of 1.37 μg mL(-1), approaching that of free DOX. Flow cytometry and confocal laser scanning microscopy studies confirmed the efficient uptake of DOX-loaded SSPEA-Gal nanoparticles by HepG2 cells as well as fast intracellular DOX release. Importantly, SSPEA-Gal and PEA-Gal nanoparticles were non-cytotoxic to HepG2 and MCF-7 cells up to a tested concentration of 1.0 mg mL(-1). These tumor-targeting and reduction-responsive degradable nanoparticles have appeared as an interesting multi-functional platform for advanced drug delivery.

Multifunctional ATRP based pH responsive polymeric nanoparticles for improved doxorubicin chemotherapy in breast cancer by proton sponge effect/endo-lysosomal escape

Folic acid and trastuzumab functionalized pH responsive polymeric nanoparticles for intracellular doxorubicin delivery in breast cancer.

Gated Mesoporous Silica Nanoparticles Using a Double‐Role Circular Peptide for the Controlled and Target‐Preferential Release of Doxorubicin in CXCR4‐Expresing Lymphoma Cells

B‐cell non‐Hodgkin's lymphoma (B‐NHL) is the most frequent malignant lymphoid neoplasm, which has a high degree of relapse and chemoresistance. Thus, strategies to improve currently used therapies are needed. In this context, a new CXCR4‐targeted delivery system is described using mesoporous silica nanoparticles (MSNs) that are loaded with doxorubicin and capped with a derivative of the T22 peptide (P). This design makes full use of the great affinity of the T22 peptide to CXCR4 receptor, which is overexpressed in lymphoma cells. The peptide is able to guide the gated nanoparticle to B‐NHL cells to facilitate MSNs uptake via the CXCR4 receptor. The endocyted P‐capped MSNs are also opened by endosomal proteolytic enzymes to allow intracellular doxorubicin delivery.

PEG-b-PCL copolymer micelles with the ability of pH-controlled negative-to-positive charge reversal for intracellular delivery of doxorubicin.

The application of PEG-b-PCL micelles was dampened by their inherent low drug-loading capability and relatively poor cell uptake efficiency. In this study, a series of novel PEG-b-PCL copolymers methoxy poly(ethylene glycol)-b-poly(ε-caprolactone-co-γ-dimethyl maleamidic acid -ε-caprolactone) (mPEG-b-P(CL-co-DCL)) bearing different amounts of acid-labile β-carboxylic amides on the polyester moiety were synthesized. The chain structure and chemical composition of copolymers were characterized by (1)H NMR, Fourier transform infrared spectroscopy (FT-IR), and gel permeation chromatography (GPC). mPEG-b-P(CL-co-DCL) with critical micellar concentrations (CMCs) of 3.2-6.3 μg/mL could self-assemble into stable micelles in water with diameters of 100 to 150 nm. Doxorubicin (DOX), a cationic hydrophobic drug, was successfully encapsulated into the polymer micelles, achieving a very high loading content due to electrostatic interaction. Then the stability, charge-conversional behavior, loading and release profiles, cellular uptake and in vitro cytotoxicity of free drug and drug-loaded micelles were evaluated. The β-carboxylic amides functionalized polymer micelles are negatively charged and stable in neutral solution but quickly become positively charged at pH 6.0, due to the hydrolysis of β-carboxylic amides in acidic conditions. The pH-triggered negative-to-positive charge reversal not only resulted in a very fast drug release in acidic conditions, but also effectively enhanced the cellular uptake by electrostatic absorptive endocytosis. The MTT assay demonstrated that mPEG-b-P(CL-co-DCL) micelles were biocompatible to HepG2 cells while DOX-loaded micelles showed significant cytotoxicity. In sum, the introduction of acid-labile β-carboxylic amides on the polyester block in mPEG-b-P(CL-co-DCL) exhibited great potentials for the modifications in the stability in blood circulation, drug solubilization, and release properties, as well as cell internalization and intracellular drug release.

Hepatocellular carcinoma dually-targeted nanoparticles for reduction triggered intracellular delivery of doxorubicin

Hepatocellular carcinoma (HCC) dual targeted stimuli responsive nanoparticles (NPs) for intracellular delivery of doxorubicin (DOX) were developed based on a reduction cleavable hyaluronic acid–glycyrrhetinic acid conjugate (HA-Cyst-GA). HA-Cyst-GA conjugate readily formed NPs in aqueous milieu and exhibited a high drug loading capacity (33.9%). The NPs redox responsiveness evaluation showed a tendency to lose their structural integrity in response to a reductive stimulus while remaining stable at physiological conditions, and that drug release was dramatically accelerated in presence of an intracellular level of glutathione. Moreover, cellular uptake studies highlighted the affinity of hepatoma cells (HepG2) toward the NPs as compared to breast cancer cells (MDA-MB-231). HA-Cyst-GA DOX-NPs displayed an increased cytotoxic potency over their non-responsive counterparts and free DOX with IC50 of 5.75, 9.33 and 10.23μg/mL, respectively. CLSM observations showed that HA-Cyst-GA DOX-NPs mediated a faster intracellular release and nuclear delivery of DOX as compared to the insensitive control. In vivo imaging study performed on H22 tumor bearing mice revealed a selective accumulation of DiR labeled NPs in the tumor and liver upon systemic administration. The antitumor efficacy was evaluated in HepG2 tumor xenograft model. Overall HA-Cyst-GA NPs appear as a potential HCC targeted intracellular delivery platform for DOX.

Simultaneous active intracellular delivery of doxorubicin and C6-ceramide shifts the additive/antagonistic drug interaction of non-encapsulated combination

Drug resistance remains the Achilles tendon undermining the success of chemotherapy. It has been recognized that success requires the identification of compounds that, when combined, lead to synergistic tumor inhibition while simultaneously minimizing systemic toxicity. However, in vivo application of such protocols is dependent on the ability to deliver the appropriate drug ratio at the tumor level. In this respect, nanotechnology-based delivery platforms, like liposomes, offer an elegant solution for the in vivo translation of such strategy.In this work, we propose the active intracellular delivery of combinations of doxorubicin and the pro-apoptotic sphingolipid, C6-ceramide, using our previously described cytosolic triggered release-enabling liposomes, targeting nucleolin with the F3 peptide.Combination of doxorubicin (DXR):C6-ceramide (C6-Cer) at 1:2 molar ratio interacted synergistically against drug resistant/triple negative MDA-MB-231 breast cancer cells, as well as drug sensitive MDA-MB-435S melanoma cells. Cell viability studies indicated that F3-targeted liposomes encapsulating DXR:C6-Cer 1:2 molar ratio (p[F3]DC12) performed similarly as targeted liposomal DXR (p[F3]SL), encapsulating twice the amount of DXR, at the IC50, for an incubation time of 24h. Importantly, F3-targeted liposomes encapsulating DXR:C6-Cer 1:2 molar ratio (p[F3]DC12) enabled a cell death above 90% at 24h of treatment against both DXR-resistant and sensitive cells, unattainable by the F3-targeted liposomal doxorubicin. Furthermore, a F3-targeted formulation encapsulating a mildly additive/antagonistic DXR:C6-Cer 1:1 molar ratio (p[F3]DC11) enabled an effect above 90% for an incubation period as short as 4h, suggesting that the delivery route at the cell level may shift the nature of drug interaction. Such activity, including the one for p[F3]DC12, induced a marked cell and nucleus swelling at similar extent, consistent with necrotic cell death.Overall, these results demonstrated that F3-targeted intracellular delivery of different DXR/C6-Cer ratios, with diversed drug interactions, enabled a highly relevant increased efficacy against chemotherapy resistant cells.

Amphiphilic hyaluronic acid derivative with the bioreducible bond: Synthesis and its implication for intracellular drug delivery

Since most of the anticancer drugs exert their action in the intracellular environment, it is imperative to deliver them selectively inside the cancer cells for effective therapy. In an attempt to develop the potential carrier for intracellular drug delivery, we prepared the amphiphilic hyaluronic acid conjugate (HA-SS-DDT) bearing the disulfide bond which could be selectively cleaved at the intracellular environment. Owing to its amphiphilicity, the HA-SS-DDT conjugate formed self-assembled nanoparticles in an aqueous condition. The nanoparticles were stable under the physiological condition (pH 7.4, 37 °C) for at least five days. Doxorubicin (DOX), chosen as the model anticancer drug, was effectively encapsulated into HA-SS-DDT nanoparticles with high efficiency (>70%) by the oil-in-water emulsion method. In the physiological solution (PBS, pH 7.4), DOX was released from HA-SS-DDT nanoparticles in a sustained manner. Interestingly, in the presence of 10 mM glutathione, a peptide abundant in the cytoplasm of the cancer cell, the release rate of DOX remarkably increased, which was due to disintegration of nanoparticles by the cleavage of the disulfide bond. DOX-loaded HA-SS-DDT (HA-SS-DDT-DOX) nanoparticles were efficiently taken up by squamous cell carcinoma (SCC7) cells via receptor-mediated endocytosis, followed by rapid release of DOX in the cytoplasm of the cells. Overall, these results indicate that HA-SS-DDT nanoparticles have promising potential as the carrier for intracellular DOX delivery.

Lipo-Poly(L-histidine) Hybrid Materials with pH-Sensitivity, Intracellular Delivery Efficiency, and Intrinsic Targetability to Cancer Cells

Biocompatible lipo-histidine hybrid materials conjugated with IR820 dye show pH-sensitivity, efficient intracellular delivery of doxorubicin (Dox), and intrinsic targetability to cancer cells. These new materials form highly uniform Dox-loaded nanosized vesicles via a self-assembly process showing good stability under physiological conditions. The Dox-loaded micelles are effective for suppressing MCF-7 tumors, as demonstrated in vitro and in vivo. The combined mechanisms of the EPR effect, active internalization, endosomal-triggered release, and drug escape from endosomes, and a long blood circulation time, clearly prove that the IR820 lipopeptide DDS is a safe theranostic agent for imaging-guided cancer therapy.

PH-sensitive pullulan-based nanoparticles for intracellular drug delivery

This study reports the design of a novel pH-sensitive nanoparticle carrier based on pullulan for realizing intracellular drug delivery. A series of pullulan derivatives (UCPA) were synthesized by conjugation of both urocanic acid (a pH-sensitive grafted moiety) and cholesterol succinate (a hydrophobically modified moiety) to pullulan. UCPAs exhibited amphiphilic and pH-sensitive properties, and their responding pH value was around 6.5. UCPA nanoparticles prepared by the precipitation method had roughly spherical shapes, and sizes ranging from 150 to 300 nm. Doxorubicin (DOX), as a model antitumor drug, was physically loaded into the UCPA nanoparticles and its in vitro release at different pH values was studied using a dialysis method. UCPA-1 nanoparticles, with the degree of substitution (DS) of urocanyl and cholesterol moieties of 6.8% and 3.5%, respectively, exhibited relatively high drug-loading capability and strong in vitro pH-induced drug release. The results of MTT assays, flow cytometric analyses and confocal microscopy observations confirmed that the UCPA-1 nanoparticles can realize the intracellular delivery of DOX after internalization and enhanced cytotoxicity of DOX against MCF-7 cells.

Biostable and bioreducible polymersomes for intracellular delivery of doxorubicin

To minimize the premature drug release of nanocarriers, we have developed chemically cross-linked bioreducible polymersomes (CLPMs) that can specifically release the drug inside cancer cells.

Novel reduction-sensitive pullulan-based micelles with good hemocompatibility for efficient intracellular doxorubicin delivery

A novel reduction-sensitive pullulan-based biocompatible material can self-assemble into nanomicelles and release loaded drug triggered by reductive condition.

A bubble-generating liposome system for intracellular delivery of doxorubicin

A bubble-generating liposome system for intracellular delivery of doxorubicin

Folate-modified poly(2-ethyl-2-oxazoline) as hydrophilic corona in polymeric micelles for enhanced intracellular doxorubicin delivery

The transmembrane transport of drug loaded micelles to intracellular compartment is quite crucial for efficient drug delivery. In the current study, we investigated the cellular internalization and anticancer activity of doxorubicin loaded micelles with folate modified stealthy PEOz corona. Folate-decorated micelles incorporating doxorubicin were characterized for particle size, degree of folate decoration, drug loading content and encapsulation efficiency, morphology, and surface charge. The targeting capability and cell viability were assessed using HeLa, KB, A549 and MCF-7/ADR cell lines. In vitro study clearly illustrated the folate receptor (FR) mediated targeting of FA modified micelles to FR-positive human HeLa, KB and MCF-7/ADR cells, while specific delivery to FR-negative A549 cells was not apparently increased at the same experimental conditions. Cytotoxicity assay showed 60% and 58% decrease in IC50 values for HeLa and KB cells, while only a slight decrease for A549 cells, following treatment with folate modified formulations. The enhanced intracellular delivery of FA modified micelles in MCF-7/ADR cells was also observed. In vivo antitumor tests revealed DOX entrapped FA-PEOz-PCL micelles effectively inhibited the tumor growth and reduced the toxicity to mice compared with free DOX. The current study showed that the targeted nano-vector improved cytotoxicity of DOX and suggested that this novel PEOz endowed stealthy micelle system held great promise in tumor targeted therapy.

Reduction/pH dual-sensitive PEGylated hyaluronan nanoparticles for targeted doxorubicin delivery

To minimize the side effect of chemotherapy, a novel reduction/pH dual-sensitive drug nanocarrier, based on PEGylated dithiodipropionate dihydrazide (TPH)-modified hyaluronic acid (PEG-SS-HA copolymer), was developed for targeted delivery of doxorubicin (DOX) to hepatocellular carcinoma. The copolymer was synthesized by reductive amination via Schiff's base formation between TPH-modified HA and galactosamine-conjugated poly(ethylene glycol) aldehyde/methoxy poly(ethylene glycol) aldehyde. Conjugation of DOX to PEG-SS-HA copolymer was accomplished through the hydrazone linkage formed between DOX and PEG-SS-HA, and confirmed by FTIR and 1H NMR spectra. The polymer–DOX conjugate could self-assemble into spherical nanoparticles (∼150nm), as indicated by TEM and DLS. In vitro release studies showed that the DOX-loaded nanoparticles could release DOX rapidly under the intracellular levels of pH and glutathiose. Cellular uptake experiments demonstrated that the nanoparticles could be efficiently internalized by HepG2 cells. These results indicate that the PEG-SS-HA copolymer holds great potential for targeted intracellular delivery of DOX.

Photo-cross-linked and pH-Sensitive Biodegradable Micelles for Doxorubicin Delivery

Cross-linked polymeric micelles have gained increasing research interest in the past decade due to the instability of existing polymeric micelles when used in vivo. In this study, we reported a series of covalently cross-linked pH-sensitive biodegradable micelles based on the poly(ethylene glycol)-hyperbranched poly(β-aminoester)s with acrylate group terminals (PEG-HBPAE-A) copolymers for intracellular delivery of doxorubicin (DOX). PEG-HBPAE-A can be self-assembled to form micellar nanoparticles in aqueous solution with diameters of approximately 160 nm. The non-cross-linked micelles (NCLMs) were cross-linked upon UV irradiation to form cross-linked micelles (CLMs). (1)H NMR, FT-IR and dynamic light scattering (DLS) were utilized to investigate the process of the UV cross-linking and the stability of CLMs. The results showed the significantly enhanced stability for CLMs in comparison to NCLMs. pH sensitivity of CLMs and NCLMs were also estimated by DLS. In vitro drug release studies confirmed that DOX release from DOX-loaded CLMs was greatly inhibited upon the neutral pH environment, whereas DOX underwent faster release in acidic conditions. MTT assays showed that DOX-loaded micelles had a similar inhibition rate for HepG-2 and MCF-7 cell lines compared with free DOX, whereas the blank CLMs and NCLMs showed very low cytotoxicity. Laser scanning confocal microscopy and real-time in situ fluorescence microscopy were exploited to investigate drug uptake in cells and drug distribution in the interior of cells. These results showed a promising nanocarrier for intracellular DOX delivery with great potential for cancer therapy.

Dual Functional Gd(III)-DOTA Liposomes for Cancer Therapy and Diagnosis as a Theragnostic Carrier

Development of dual functional liposome has been studied for cancer theragnostics. Therefore, we focused on ultrasound-sensitive liposomes with doxorubicin (DOX) and gadolinium (Gd) as a theragnostic carrier having a potential for cancer therapy and diagnosis. In this study, Gd(III)-DOTA-modified sonosensitive liposomes (GL) was developed using chemically synthesized Gd(III)-DOTA-DPPE lipid. Sonosensitivity of GL to 1 MHz ultrasound induced 25% of DOX release. The relaxivities () of GL were , which was higher than that of MR-bester. Intracellular delivery of DOX from GL by ultrasound irradiation was evaluated according to ultrasound intensity, resulting in increase of uptake of DOX released from ultrasound-triggered GLs compared to GL3 or Doxil without ultrasound. Taken together, this study shows that the paramagnetic and sonosensitive liposomes, GL, is a novel and highly effective delivery system for drug with the potential for broad applications in human disease.

Study of Intracellular Delivery of Doxorubicin from Poly(lactide‐co‐glycolide) Nanoparticles by Means of Fluorescence Lifetime Imaging and Confocal Raman Microscopy

The intracellular delivery of Doxorubicin (Dox) from poly(lactide-co-glycolide) (PLGA) nanoparticles stabilised with bovine serum albumin, in HepG2 cells, is studied via flow cytometry, fluorescence lifetime imaging microscopy (FLIM), confocal Raman microscopy (CRM) and cell viability studies. Flow cytometry shows that the initial uptake of PLGA and Dox follow the same kinetics. However, following 8 h of incubation, the fluorescence intensity and cellular uptake of Dox decreases, while in the case of PLGA both parameters remain constant. FLIM shows the presence of a single-lifetime species, with a lifetime of 1.15 ns when measured inside the cells. Cell viability decreases by approximately 20% when incubated for 24 h with PLGA loaded with Dox, with a particle concentration of 100 µg · mL(-1). At the single-cell level, CRM shows changes in the bands from DNA and proteins in the cell nucleus when incubated with PLGA loaded with Dox.

Intracellular delivery of doxorubicin encapsulated in novel pH-responsive chitosan/heparin nanocapsules

A novel polyelectrolyte nanocapsule system composed of biopolymers, chitosan and heparin has been fabricated by the layer-by-layer technique on silica nanoparticles followed by dissolution of the silica core. The nanocapsules were of the size range 200 ± 20 nm and loaded with the positively charged anticancer drug doxorubicin with an efficiency of 89%. The loading of the drug into the capsule happens by virtue of the pH-responsive property of the capsule wall, which is determined by the pKa of the polyelectrolytes. As the pH is varied, about 64% of the drug is released in acidic pH while 77% is released in neutral pH. The biocompatibility, efficiency of drug loading, and enhanced bioavailability of the capsule system was confirmed by MTT assay and in vivo biodistribution studies.