Analysis of the effect of HDAC inhibitors on the formation of the HIV reservoir.

The HIV reservoir is more dynamic than previously thought with around 70% of the latent reservoir originating from viruses circulating within 1 year of the initiation of antiretroviral therapy (ART). In an ex vivo model system of HIV latency, it was reported that early exposure to class I histone deacetylase (HDAC) inhibitors might prevent these more recently infected cells from entering a state of stable viral latency. This finding raises the possibility that co-administration of HDAC inhibitors at the time of ART initiation may prevent the establishment of much of the HIV reservoir. Here, we tested the effects of the HDAC inhibitors suberoylanilide hydroxamic acid (SAHA) and panobinostat co-administered at the time of ART initiation on the formation of the viral reservoir in HIV-infected humanized mice. As previously shown, SAHA and panobinostat were well tolerated in humanized mice. Unexpectedly, co-administration of SAHA resulted in an increase in the frequency of CD4+ cells carrying HIV DNA but did not alter the frequency of cell-associated HIV RNA in HIV-infected, ART-treated humanized mice. Co-administration of panobinostat did not alter levels of cell-associated HIV DNA or RNA. Our in vivo findings indicate that co-administration of HDAC inhibitors initiated at the same time of ART treatment does not prevent recently infected cells from entering latency.IMPORTANCECurrent antiretroviral therapy (ART) does not eradicate cells harboring replication-competent HIV reservoir. Withdrawal of ART inevitably results in a rapid viremia rebound. The HIV reservoir is more dynamic than previously thought. Early exposure to class I histone deacetylase (HDAC) inhibitors inhibit these more recently infected cells from entering a state of stable viral latency in an ex vivo model of latency, raising the possibility that co-administration of HDAC inhibitors at the time of ART initiation may reduce much of the HIV reservoir. Here, we tested the effects of the HDAC inhibitors suberoylanilide hydroxamic acid or panobinostat during ART initiation on the formation of the viral reservoir in HIV-infected humanized mice. Our in vivo study indicates that in contrast to in vitro observations, the co-administration of HDAC inhibitors at the same time of ART initiation does not prevent recently infected cells from entering latency.

A long-acting formulation of rifabutin is effective for prevention and treatment of Mycobacterium tuberculosis.

Tuberculosis (TB) is a communicable disease caused by Mycobacterium tuberculosis (Mtb) and is a major cause of morbidity and mortality. Successful treatment requires strict adherence to drug regimens for prolonged periods of time. Long-acting (LA) delivery systems have the potential to improve adherence. Here, we show the development of LA injectable drug formulations of the anti-TB drug rifabutin made of biodegradable polymers and biocompatible solvents that solidifies after subcutaneous injection. Addition of amphiphilic compounds increases drug solubility, allowing to significantly increase formulation drug load. Solidified implants have organized microstructures that change with formulation composition. Higher drug load results in smaller pore size that alters implant erosion and allows sustained drug release. The translational relevance of these observations in BALB/c mice is demonstrated by (1) delivering high plasma drug concentrations for 16 weeks, (2) preventing acquisition of Mtb infection, and (3) clearing acute Mtb infection from the lung and other tissues.

Targeted delivery of anti-inflammatory cytokine by nanocarrier reduces atherosclerosis in Apo E-/- mice.

Unresolved inflammation is a hallmark of many deadly diseases including atherosclerosis, a silent pathological condition behind majority of cardiovascular diseases. Yet, anti-inflammatory drugs are not clinically used in the treatment of patients with atherosclerosis. The currently approved treatment regimen against atherosclerosis is mainly focused on lowering the cholesterol/lipid levels in blood and has little to do with controlling inflammation, the underlying cause. Recent preclinical and clinical data suggest that effective alleviation of inflammation in the atherosclerosis plaque could reduce the risk of cardiovascular disease. In this work, we have encapsulated interleukin-10 (IL10), a multipotent anti-inflammatory cytokine into cRGD conjugated pluronic based nano-carriers (NC) for targeted delivery to atherosclerotic plaques. The NC could encapsulate the therapeutic protein with a high loading efficiency in a mild condition and showed sustained release capabilities. The efficacy of cytokine encapsulated NC was analyzed in vitro using the lipopolysaccharide stimulated macrophage cells and in vivo using an established apolipoprotein E-knockout (ApoE-/-) C57BL/6 mouse model. Compared to free IL10, intravenous administration of NC encapsulated IL10 resulted in vastly improved pharmacokinetic profile and profoundly high accumulation of the cytokine in the atherosclerosis lesions. IL10 delivered by NC was bioactive and reduced the production of pro-inflammatory cytokine IL-1ß in the lesion and led to significant regression in the plaque size. These results signify the prospect of nanoparticle based cytokine delivery for preventing atherosclerotic through inflammation modulation in near future.

Co-delivery of therapeutic protein and catalase-mimic nanoparticle using a biocompatible nanocarrier for enhanced therapeutic effect.

Therapeutic proteins are indispensable in the treatment of various human diseases. Despite the many benefits of therapeutic proteins, they also exhibit diverse side effects. Therefore, reducing unwanted side effects of therapeutic proteins as well as enhancing their therapeutic efficacy are very important in developing therapeutic proteins. Urate oxidase (UOX) is a therapeutic enzyme that catalyzes the conversion of uric acid (UA) into a soluble metabolite, and it is used clinically for the treatment of hyperuricemia. Since UA degradation by UOX generates H2O2 (a cytotoxic side product), UOX was co-delivered with catalase-mimic nanoparticles (AuNPs) using biocompatible pluronic-based nanocarriers (NCs) to effectively reduce H2O2-associated toxicity in cultured cells and to enhance UA degradation efficiency in vivo. Simple temperature-dependent size changes of NCs allowed co-encapsulation of both UOX and AuNPs at a high loading efficiency without compromising critical properties, resulting in efficient modulation of a mixing ratio of UOX and AuNPs encapsulated in NCs. Co-localizing UOX and AuNPs in the NCs led to enhanced UA degradation and H2O2 removal in vitro, leading to a great reduction in H2O2-associated cytotoxicity compared with UOX alone or a free mixture of UOX and AuNPs. Furthermore, we demonstrated that co-delivery of UOX and AuNPs using NCs significantly improves in vivo UA degradation compared to simple co-injection of free UOX and AuNPs. More broadly, we showed that biocompatible pluronic-based nanocarriers can be used to deliver a target therapeutic protein along with its toxicity-eliminating agent in order to reduce side effects and enhance efficacy.

Comparison of in vivo targeting ability between cRGD and collagen-targeting peptide conjugated nano-carriers for atherosclerosis.

Atherosclerosis plaque is a major cause of cardiovascular diseases across the globe and a silent killer. There are no physical symptoms of the disease in its early stage and current diagnostic techniques cannot detect the small plaques effectively or safely. Plaques formed in blood vessels can cause serious clinical problems such as impaired blood flow or sudden death, regardless of their size. Thus, detecting early stage of plaques is especially more important to effectively reduce the risk of atherosclerosis. Nanoparticle based delivery systems are recognized as a promising option to fight against this disease, and various targeting ligands are typically used to improve their efficiency. So, the choice of appropriate targeting ligand is a crucial factor for optimal targeting efficiency. cRGD peptide and collagen IV targeting peptide, which binds with the αvß3 integrin overexpressed in the neovasculature of the plaque and collagen type IV present in the plaque, respectively, are frequently used for the targeting of nanoparticles. However, at present no study has directly compared these two peptides. Therefore, in this study, we have prepared cRGD or collagen IV targeting (Col IV-tg-) peptide conjugated and iron oxide nanoparticle (IONP) loaded Pluronic based nano-carriers for systemic comparison of their targeting ability towards in vivo atherosclerotic plaque in Apolipoprotein E deficient (Apo E-/-) mouse model. Nano-carriers with similar size, surface charge, and IONP loading content but with different targeting ligands were analyzed through in vitro and in vivo experiments. Near infrared fluorescence imaging and magnetic resonance imaging techniques as well as Prussian blue staining were used to compare the accumulation of different ligand conjugated nano-caariers in the aorta of atherosclerotic mice. Our results indicate that cRGD based targeting is more efficient than Col IV-tg-peptide in the early stage of atherosclerosis.

Nanographene oxide as a switch for CW/pulsed NIR laser triggered drug release from liposomes.

The application of pulsed and continuous wave (CW) lasers in nanomedicine has increased significantly over the last decade. Near infrared (NIR) lasers can be used for the precise control of drug release at the target site in a non-invasive manner. In this study, we have prepared nanographene oxide (nGO, size ~40nm) integrated liposomes (size ~900nm). The nGOs were not simply adsorbed onto the liposome surface but was embedded inside the liposomes as characterized by cryo-TEM, selected area electron diffraction (SAED), and fluorescence quenching studies. The embedded nGOs could act as a molecular switch for NIR light controlled drug release from the liposomes. Calcein was encapsulated into the liposome as a model drug to evaluate the efficiency of light controlled release. An on-demand pulsatile drug release was achieved by irradiation of CW/pulsed NIR lasers into the nGO-liposome suspension. Triggering with a pulsed laser resulted in larger release of calcein with a minimal temperature increase (~2°C) of the liposome solution, compared to lower release rate and a significant temperature increase (~8°C) by a CW laser with the same light energy, suggesting two separate mechanisms and different potential applications depending on the laser type.

Injectable system and its potential application for the delivery of biomolecules by using thermosensitive poly(γ-glutamic acid)-based physical hydrogel.

Poly (γ-glutamic acid) (PGA) is a natural biomaterial with numerous good properties such as non-toxicity, non-immunogenicity, and water holding. So, various forms of PGA-based materials have been developed for bio-applications, including sheet, cross-linked hydrogel, and nanoparticle except injectable hydrogel type. Considering inherent advantages of injectable system, injectable hydrogel based on PGA can broaden the bio-application range of PGA. In this study, a PGA-based injectable hydrogel system was prepared by physically mixing it with a small amount of chitosan with two different water solubility and molecular weight. The prepared hydrogel system showed a thermo-sensitivity through sol-gel transition behavior in the body temperature change. The mechanical properties and in vitro stability could be modulated by varying the ratio of two types of chitosan. In vitro protein (human bFGF) delivery using this injectable formulation showed a sustained release for ∼2 weeks while preserving its bioactivity. In addition, the in situ formation of this PGA-based hydrogel formulation upon subcutaneous injection was demonstrated in vivo. The hydrogel formed in vivo also showed a long term (∼2 weeks) stability without noticeable inflammatory response. Therefore, the present formulation can be applied as a promising injectable hydrogel system for tissue engineering or drug delivery.

Nanosized Ultrasound Enhanced-Contrast Agent for in Vivo Tumor Imaging via Intravenous Injection.

To enhance the detection limit of ultrasound (US) imaging, ultrasound enhanced-contrast agents (UECAs) that can go preferentially to the target tissue such as a tumor and amplify the US signal have been developed. However, nanosized UECAs among various UECAs developed are very limited to clearly demonstrate proper ability for selective tumor detection by US imaging upon their intravenous injection. In this study, we prepared CaCO3 nanoparticles that were formed inside a flexible and biocompatible pluronic-based nanocarrier. This nanosized UECA was stable in serum-containing media and generated CO2, more preferentially at low pH; thus, it could be detected by US imaging. After intravenous injection into tumor-bearing mice, this nanosized UECA showed a significant US contrast enhancement at the tumor site in 1 h, in contrast to no change in the liver, followed by a rapid clearance from the body in 24 h. Therefore, the present nanosized UECA could be applied as an effective diagnostic modality for in vivo tumor imaging by ultrasonography.

The enhanced anti-tissue adhesive effect of injectable pluronic-HA hydrogel by poly(γ-glutamic acid).

The stability of tissue barrier in physiological condition is a key factor to isolate the damaged site from adjacent tissue for anti-tissue adhesion. Although pluronic or pluronic-hyaluronic acid (HA) hydrogel as an injectable formulation can prevent tissue adhesion at the injection site, the anti-tissue adhesion effect is limited due to its poor stability. Herein, we prepared tissue barrier formulations composed of pluronic F127 (F127) and HA mixture (F127-HA) and the effect of the addition of poly(γ-glutamic acid) (PGA) was characterized. All of F127, HA, and F127-HA mixture showed the poor in vitro residence stability less than 3 days. However, by adding PGA into F127-HA mixture, their stability was significantly enhanced by the control of the molecular weight and concentration of PGA. Thus, F127-HA with 10wt% PGA (2000kDa) showed the long-term stability over 10 days. Similarly, the enhanced stability of F127-HA with PGA resulted in the enhanced and excellent in vivo anti-tissue adhesion effect, evidenced by histological analysis and grading of tissue adhesion. Therefore, F127-HA containing PGA could be applied as an efficient injectable tissue barrier for anti-tissue adhesion.

Systemic modulation of the stability of pluronic hydrogel by a small amount of graphene oxide.

Thermo-sensitive and injectable hydrogels have been widely investigated for drug delivery, tissue engineering, and other biomedical applications. Pluronic copolymers can form thermo-sensitive physical gel state, thus applicable for injectable hydrogels. However, they are not stable in vivo, showing a very fast dissolution, which limits their applications. We propose a novel Pluronic-based physical hydrogel with enhanced stability by simply adding a small quantity of graphene oxide (GO) which has a large surface area and can make strong interactions with Pluronic. Further carboxylated GO could act as a more efficient additive. The addition of GO increased the moduli of hydrogels, but more importantly, it enhanced the stability of Pluronic gel dramatically. The in vitro dissolution rate of Pluronic hydrogel could be systematically modulated by increasing GO content. Upon subcutaneous injection at a sol state, GO-containing hydrogel induced a stable gel state, and was maintained over several weeks whereas very fast degradation was observed without the addition of GO. Furthermore, histological analyses demonstrated that the GO-containing Pluronic hydrogel was biocompatible and showed no severe inflammatory response. Similarly, GO-containing hydrogel resulting from the packing of Pluronic-based nanogel also showed the more enhanced stability by the addition of GO both in vitro and in vivo. In both systems, hydrogels with remarkably enhanced stability by the addition of GO were also effective for the sustained release of loaded protein, and the release rates were mainly determined by the degradation rates of hydrogels. Thus, these GO-containing Pluronic systems can be used as a thermo-sensitive injectable system with a sufficient stability in vivo.

Tumor-targeting nanogel that can function independently for both photodynamic and photothermal therapy and its synergy from the procedure of PDT followed by PTT.

A dual-function nano-system for synergistic photodynamic therapy (PDT) and photothermal therapy (PTT) was constructed. Gold nanorods (GNRs) as a PTT agent and chlorin e6 (Ce6) as a photosensitizer (PS) for PDT were loaded into a chitosan-functionalized, Pluronic-based nanogel that was proven to be an efficient delivery vehicle to the tumor site in vivo. Previously reported combined therapy systems relied on quenching and de-quenching of PS for PDT upon thermo-impact of PTT, thus only PTT followed by PDT procedure was possible. In contrast, the present dual-acting system has no quenching between PS and GNRs by preventing direct contact and self-aggregation of photo-sensitizers, allowing independent PDT or PTT procedure. In both in vitro cell culture and in vivo tumor-bearing mice experiments, a remarkably enhanced tumor ablation compared to the treatment of PDT or PTT only was observed by the treatment of PDT followed by PTT, but not significantly by the treatment of PTT followed by PDT. Thus, the present study demonstrated the synergistic effect of PDT and PTT in a sequence-dependent manner, and our system is a promising dual function nano system to achieve the enhanced phototherapy in vivo.