A FRET-guided, NIR-responsive bubble-generating liposomal system for in vivo targeted therapy with spatially and temporally precise controlled release.

The nonspecific distribution of therapeutic agents and nontargeted heating commonly produce undesirable side effects during cancer treatment since the optimal timing of triggering the carrier systems is unknown. This work proposes a multifunctional liposomal system that can intracellularly and simultaneously deliver the therapeutic drug doxorubicin (DOX), heat, and a bubble-generating agent (ammonium bicarbonate, ABC) into targeted tumor cells to have a cytotoxic effect. Gold nanocages that are encapsulated in liposomes effectively convert near-infrared light irradiation into localized heat, which causes the decomposition of ABC and generates CO2 bubbles, rapidly triggering the release of DOX. Additionally, a hybridized Mucin-1 aptamer is conjugated on the surface of the test liposomes, which then function as a recognition probe to enhance the uptake of those liposomes by cells, and as a molecular beacon to signal when the internalized particles have been maximized, which is the optimal time for photothermally triggering the release of the drug following the systemic administration of the liposomes. Empirical results reveal that this combined treatment effectively controls targeted drug release in a spatially and temporally precise fashion and so significantly increases the potency of the drug while minimizing unwanted side effects, making it a promising treatment for cancer.

An AS1411 aptamer-conjugated liposomal system containing a bubble-generating agent for tumor-specific chemotherapy that overcomes multidrug resistance.

Recent research in chemotherapy has prioritized overcoming the multidrug resistance (MDR) of cancer cells. In this work, liposomes that contain doxorubicin (DOX) and ammonium bicarbonate (ABC, a bubble-generating agent) are prepared and functionalized with an antinucleolin aptamer (AS1411 liposomes) to target DOX-resistant breast cancer cells (MCF-7/ADR), which overexpress nucleolin receptors. Free DOX and liposomes without functionalization with AS1411 (plain liposomes) were used as controls. The results of molecular dynamic simulations suggest that AS1411 functionalization may promote the affinity and specific binding of liposomes to the nucleolin receptors, enhancing their subsequent uptake by tumor cells, whereas plain liposomes enter cells with difficulty. Upon mild heating, the decomposition of ABC that is encapsulated in the liposomes enables the immediate activation of generation of CO2 bubbles, creating permeable defects in their lipid bilayers, and ultimately facilitating the swift intracellular release of DOX. In vivo studies in nude mice that bear tumors demonstrate that the active targeting of AS1411 liposomes can substantially increase the accumulation of DOX in the tumor tissues relative to free DOX or passively targeted plain liposomes, inhibiting tumor growth and reducing systemic side effects, including cardiotoxicity. The above findings indicate that liposomes that are functionalized with AS1411 represent an attractive therapeutic alternative for overcoming the MDR effect, and support a potentially effective strategy for cancer therapy.

Hyperthermia-mediated local drug delivery by a bubble-generating liposomal system for tumor-specific chemotherapy.

As is widely suspected, lysolipid dissociation from liposomes contributes to the intravenous instability of ThermoDox (lysolipid liposomes), thereby impeding its antitumor efficacy. This work evaluates the feasibility of a thermoresponsive bubble-generating liposomal system without lysolipids for tumor-specific chemotherapy. The key component in this liposomal formulation is its encapsulated ammonium bicarbonate (ABC), which is used to actively load doxorubicin (DOX) into liposomes and trigger a drug release when heated locally. Incubating ABC liposomes with whole blood results in a significantly smaller decrease in the retention of encapsulated DOX than that by lysolipid liposomes, indicating superior plasma stability. Biodistribution analysis results indicate that the ABC formulation circulates longer than its lysolipid counterpart. Following the injection of ABC liposome suspension into mice with tumors heated locally, decomposition of the ABC encapsulated in liposomes facilitates the immediate thermal activation of CO2 bubble generation, subsequently increasing the intratumoral DOX accumulation. Consequently, the antitumor efficacy of the ABC liposomes is superior to that of their lysolipid counterparts. Results of this study demonstrate that this thermoresponsive bubble-generating liposomal system is a highly promising carrier for tumor-specific chemotherapy, especially for local drug delivery mediated at hyperthermic temperatures.

The use of MMP2 antibody-conjugated cationic microbubble to target the ischemic myocardium, enhance Timp3 gene transfection and improve cardiac function.

The objective of this study was to synthesize a cationic microbubble (CMB) conjugated with an antibody against matrix metalloproteinase 2 (CMBMMP2) to increase microbubble accumulation and gene transfection in the infarcted myocardium and to restore ventricular function following an ischemic insult. We previously reported that our CMBs enhanced the efficiency of gene transfection following ultrasound-targeted microbubble destruction (UTMD) in rodent hearts. Therefore, we conjugated a thiolated MMP2 antibody to the PEG chains on the CMB surface, which was verified by fluorescent microscopy. Rats underwent ischemia/reperfusion injury 3 days prior to UTMD delivery of the control or Timp3 plasmid. The CMBMMP2 improved microbubble accumulation in the infarct region, with 57% more contrast intensity compared to the non-conjugated CMB. UTMD-mediated CMBMMP2 delivery of the Timp3 gene significantly increased TIMP3 protein levels in the infarct scar and border zone at 3 days post-UTMD compared to delivery by the non-conjugated CMB. Both MMP2 and MMP9 activity were reduced in the CMBMMP2Timp3 group, which resulted in smaller and thicker infarcts and improved cardiac function. UTMD therapy with this CMBMMP2 provides an efficient platform for the targeted delivery of factors intended to preserve ventricular structure and improve cardiac function after ischemic injury.

A thermoresponsive bubble-generating liposomal system for triggering localized extracellular drug delivery.

The therapeutic effectiveness of chemotherapy is optimal only when tumor cells are subjected to a maximum drug exposure. To increase the intratumoral drug concentration and thus the efficacy of chemotherapy, a thermoresponsive bubble-generating liposomal system is proposed for triggering localized extracellular drug delivery. The key component of this liposomal formulation is the encapsulated ammonium bicarbonate (ABC), which is used to create the transmembrane gradient needed for a highly efficient encapsulation of doxorubicin (DOX). At an elevated temperature (42 °C), decomposition of ABC generates CO(2) bubbles, creating permeable defects in the lipid bilayer that rapidly release DOX and instantly increase the drug concentration locally. Because the generated CO(2) bubbles are hyperechogenic, they also enhance ultrasound imaging. Consequently, this new liposomal system encapsulated with ABC may also provide an ability to monitor a temperature-controlled drug delivery process.

Three-dimensional cell aggregates composed of HUVECs and cbMSCs for therapeutic neovascularization in a mouse model of hindlimb ischemia.

The proximity of cells in three-dimensional (3D) organization maximizes the cell-cell communication and signaling that are critical for cell function. In this study, 3D cell aggregates composed of human umbilical vein endothelial cells (HUVECs) and cord-blood mesenchymal stem cells (cbMSCs) were used for therapeutic neovascularization to rescue tissues from critical limb ischemia. Within the cell aggregates, homogeneously mixed HUVECs and cbMSCs had direct cell-cell contact with expressions of endogenous extracellular matrices and adhesion molecules. Although dissociated HUVECs/cbMSCs initially formed tubular structures on Matrigel, the grown tubular network substantially regressed over time. Conversely, 3D HUVEC/cbMSC aggregates seeded on Matrigel exhibited an extensive tubular network that continued to expand without regression. Immunostaining experiments show that, by differentiating into smooth muscle cell (SMC) lineages, the cbMSCs stabilize the HUVEC-derived tubular network. The real-time PCR analysis results suggest that, through myocardin, TGF-ß signaling regulates the differentiation of cbMSCs into SMCs. Transplantation of 3D HUVEC/cbMSC aggregates recovered blood perfusion in a mouse model of hindlimb ischemia more effectively compared to their dissociated counterparts. The experimental results confirm that the transplanted 3D HUVEC/cbMSC aggregates enhanced functional vessel formation within the ischemic limb and protected it from degeneration. The 3D HUVEC/cbMSC aggregates can therefore facilitate the cell-based therapeutic strategies for modulating postnatal neovascularization.

The use of cationic microbubbles to improve ultrasound-targeted gene delivery to the ischemic myocardium.

We synthesized a cationic microbubble (CMB) with the aim of enhancing its DNA-carrying capacity to improve targeted gene transfection of the ischemic heart for cardiac regeneration. We previously reported that ultrasound-targeted microbubble destruction (UTMD) employing the commercial Definity microbubble (MB) successfully transfected genes into rodent hearts, but the transfection efficiency was modest. We synthesized a CMB and compared its DNA-carrying capacity and reporter gene transfection efficiency with the Definity MB. The CMB bound 70% more plasmid DNA than the Definity MB. UTMD-mediated gene delivery with the CMB enhanced both transfection efficiency and gene expression. In vivo studies assessed the ability of the CMB to deliver the therapeutic AKT gene to the ischemic rat myocardium and evaluated the effects on apoptosis, angiogenesis, and cardiac function. AKT transfection with the CMB reduced infarct size (p < 0.05), increased infarct thickness (p < 0.05), reduced apoptosis (p < 0.05), increased vascular density (p < 0.05), and improved cardiac perfusion and function (p < 0.05) compared to the Definity MB. Delivery of AKT with the CMB resulted in greater cardiac functional improvements compared to the Definity MB. UTMD therapy with this CMB provides an efficient platform for the targeted delivery of factors required to regenerate the ischemic heart and preserve cardiac function.

Multidrug release based on microneedle arrays filled with pH-responsive PLGA hollow microspheres.

This work presents an approach to codelivering transdermally two model drugs, Alexa 488 and Cy5, in sequence, based on a system of polyvinylpyrrolidone microneedles (PVP MNs) that contain pH-responsive poly(d,l-lactic-co-glycolic acid) hollow microspheres (PLGA HMs). The MN system provides the green fluorescence of Alexa 488 in PVP MNs, the red fluorescence of the DiI-labeled PLGA shell of HMs, and the cyan fluorescence of Cy5 in their aqueous core. Combined together, the prepared MN arrays support the localization of the HMs and the monitoring of the release profiles of model drugs within the skin tissues. The key component of this system is NaHCO(3), which can be easily incorporated into HMs. After HMs are treated with an acidic solution (simulating the skin pH environment), protons (H(+)) can rapidly diffuse through the free volume in the PLGA shells to react with NaHCO(3) and form a large number of CO(2) bubbles. This effect generates pressure inside the HMs and creates pores inside their PLGA shells, releasing the encapsulated Cy5. Test MNs were strong enough to be inserted into rat skin without breaking. The PVP MNs were significantly dissolved within minutes, and the first model drug Alexa 488, together with HMs, were successfully deposited into the tissues. Once in the acidic environment of the skin, the released HMs started to release Cy5 and continued to spread throughout the neighboring tissues, in a second step of the release of the drug. This approach can be used clinically to codeliver sequentially and transcutaneously a broad range of drugs.

A review of the prospects for polymeric nanoparticle platforms in oral insulin delivery.

Success in the oral delivery of therapeutic insulin can significantly improve the quality of life of diabetic patients who must routinely receive injections of this drug. However, oral absorption of insulin is limited by various physiological barriers and remains a major scientific challenge. Various technological solutions have been developed to increase the oral bioavailability of insulin. Having received considerable attention, nano-sized polymeric particles are highly promising for oral insulin delivery. This review article describes the gastrointestinal barriers to oral insulin delivery, including chemical, enzymatic and absorption barriers. The potential transport mechanisms of insulin delivered by nanoparticles across the intestinal epithelium are also discussed. Finally, recent advances in using polymeric nanoparticles for oral insulin delivery and their effects on insulin transport are reviewed, along with their future.

Intracellularly monitoring/imaging the release of doxorubicin from pH-responsive nanoparticles using Förster resonance energy transfer.

Stimuli-responsive nanoparticles (NPs) have been receiving much attention as a drug-delivery vehicle for therapeutic applications; once internalized into cells, the intracellular fate of NPs and their drug release behavior in response to local stimuli must be understood for efficient delivery of therapeutics. In this study, we prepared pH-responsive doxorubicin (DOX)-loaded NPs, made of N-palmitoyl chitosan bearing a Cy5 moiety (Cy5-NPCS), as an anticancer delivery device. The results of our molecular dynamic simulations showed that the ability of Cy5-NPCS to self-associate offered the close proximity between the donor (DOX) and the acceptor (Cy5) required for Förster resonance energy transfer (FRET), while the pH-driven structure transition prescribed the on-to-off switch of the energy transfer. The caveolae-mediated pathway played a major role in the internalization of NPCS NPs. Using the concept of FRET, we found that the DOX fluorescence in the cytosol was first seen when NPCS NPs were present in the slightly acidic early endosomes. Following NPCS NPs trafficking into a more acidic organelle (late endosomes/lysosomes), a more evident release of DOX into the cytosol was observed; the released DOX was then gradually accumulated in the cell nuclei, leading to a significant cytotoxicity. Understanding the fate of NPs with respect to their intracellular localization and drug release behavior is crucial for the rational design of drug carriers.

A dual-emission Förster resonance energy transfer nanoprobe for sensing/imaging pH changes in the biological environment.

A dual-emission nanoprobe that can sense changes in the environmental pH is designed based on the concept of pH-responsive Förster resonance energy transfer induced by the conformational transition of an associating polyelectrolyte, N-palmitoyl chitosan, bearing a donor (Cy3) or an acceptor (Cy5) moiety. We demonstrate that the developed pH-responsive nanoprobe can be used to ratiometrically image and thus discriminate the pH changes in the biological environment at different length scales.

The characteristics, cellular uptake and intracellular trafficking of nanoparticles made of hydrophobically-modified chitosan.

It has been reported that nanoparticles (NPs) prepared by hydrophobically-modified polymers could accumulate passively in the tumor tissue; however, their cellular uptake mechanism and intercellular trafficking pathway have never been understood. This study was designed to address these concerns, using NPs prepared by a hydrophobically-modified chitosan (N-palmitoyl chitosan, NPCS). Molecular dynamic simulations found that a degree of substitution (DS) of 5% of palmitoyl groups on its backbone was sufficient to allow NPCS to form NPs, due to a significant increase in the intra- and intermolecular hydrophobic interactions. With an increase of DS, there were more palmitoyl groups present on the surface of NPs which were then able to interact with the cell membranes. A greater extent of cellular uptake of NPCS NPs was observed with increasing the DS on NPCS. The internalization of NPCS NPs was clearly related with the lipid raft-mediated routes; with increasing the DS on NPCS, the caveolae-mediated endocytosis became more important. The results obtained in the intracellular trafficking study showed that NPCS NPs entered cells via caveolae and transiently localized to caveosomes before trafficking to the endosomal pathway. These results suggest that the prepared NCPS NPs may serve as a carrier for intracellular delivery of therapeutic agents.