Doxorubicin-loaded nanoparticles consisted of cationic- and mannose-modified-albumins for dual-targeting in brain tumors.

Albumin nanoparticles have been increasingly viewed as an effective way of delivering chemotherapeutics to solid tumors. Here, we report the one-pot development of a unique prototype of doxorubicin-loaded nanoparticles (NPs) made of naïve albumin (HSA) plus cationic- (c-HSA) or mannose-modified-albumin (m-HSA), with the goal of traversing the blood-brain barrier and targeting brain tumors. c-HSA was synthesized by conjugating ethylenediamine to naïve HSA. Then, m-HSA was derivatized using mannopyranoside via a thiol-maleimide reaction. The c/m-HSA NPs were prepared using a mixture solution of c- and m-HSAs in deionized water and doxorubicin in ethanol/chloroform in the same pot using a high-pressure homogenizer. The c/m-HSA NPs were spherical and well-dispersed, with a particle size of 90.5±3.1nm and zeta-potential of -12.0±0.3mV at c- and m-HSA feed ratios of 5% and 10%, respectively. The c/m-HSA NPs displayed good stability over 3days based on particle size and a linear gradual doxorubicin release over 2days. Specifically, the inhibitory concentration (IC50; 0.5±0.02µg/ml) of c/m-HSA NPs was >2.2-15.6 fold lower than those of doxorubicin or the other HSA NPs. Moreover, among HSA NPs, c/m-HSA NPs exhibited the most prominent performances in transport across the bEnd.3 cell monolayer and uptake in bEnd.3 cells as well as U87MG glioblastoma cells and spheroids. Furthermore, c/m-HSA NPs were localized to a greater extent in brain glioma compared to naïve HSA NPs. Orthotopic glioma-bearing mice treated with c/m-HSA NPs displayed significantly smaller tumors than the mice treated with saline, doxorubicin or HSA NPs. This improved anti-glioma efficacy seemed to be due to the dual-enhanced system of dual cationic absorptive transcytosis and glucose-transport by the combined use of c- and m-HSAs. The c/m-HSA NPs have potential as a novel anti-brain cancer agent with good targetability.

Doxorubicin-Bound Albumin Nanoparticles Containing a TRAIL Protein for Targeted Treatment of Colon Cancer.

PURPOSE: We developed a new nanoparticle formulation comprised of human serum albumin (HSA) for co-delivery of doxorubicin (Dox) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with the goal of apoptotic synergy in the treatment of colon cancer. METHODS: TRAIL (0.2, 0.4, 1.0%)- and Dox-loaded HSA nanoparticles (TRAIL/Dox HSA NPs) were prepared by using the nab(TM) technology. Morphological and physicochemical characterizations were investigated by dynamic light scattering and transmission electron microscopy. Synergistic cytotoxicity, apoptotic activity, and potential penetration into mass tumor were determined in HCT116 cell-based systems. Furthermore, antitumor efficacy and tumor targeting were also investigated. RESULTS: TRAIL/Dox HSA NPs were uniformly spherical with sizes of 60 ~ 120 nm. The encapsulation efficacy of Dox and TRAIL was 68.9-77.2% and 80.4-86.0%, respectively. TRAIL 1.0%/Dox HSA NPs displayed the best inhibition of HCT116 colon cancer cells; inhibition was 6 times higher than achieved with Dox HSA NPs. The TRAIL 1.0%/Dox HSA NPs formulation was studied further. Flow cytometry analysis and TUNEL assay revealed that TRAIL 1.0%/Dox HSA NPs had markedly greater apoptotic activity than Dox HSA NPs. In HCT116 tumor-bearing BALB/c nu/nu mice, TRAIL 1.0%/Dox HSA NPs had significantly higher antitumor efficacy than Dox HSA NPs (tumor volume; 933.4 mm(3) vs. 3183.7 mm(3), respectively). TRAIL 1.0%/Dox HSA NPs penetrated deeply into tumor masses in a HCT116 spheroid model and localized in tumor sites after tail vein injection. CONCLUSIONS: Data indicate that TRAIL 1.0%/Dox HSA NPs offer advantages of co-delivery of Dox and TRAIL in tumors, with potential synergistic apoptosis-based anticancer therapy.

Pharmaceutical potential of tacrolimus-loaded albumin nanoparticles having targetability to rheumatoid arthritis tissues.

Albumin is considered an attractive dug carrier for hydrophobic drugs to target inflamed joints of rheumatoid arthritis. This study focused on the pharmaceutical potential of albumin-based nanoparticles (NPs) on delivery of tacrolimus (TAC) to enhance targetability and anti-arthritic efficacy. TAC-loaded human serum albumin (HSA) nanoparticles (TAC HSA-NPs) were prepared using the nab™ technology. The resulting NPs were 185.8 ± 6.8 nm in diameter and had a zeta potential value of -30.5 ± 1.1 mV, as determined by dynamic light scattering. Particles were uniformly spherical in shape as determined by transmission electron microscopy. The encapsulation efficacy of TAC was 79.3 ± 3.7% and the water solubility was over 46 times greater than that of free TAC. TAC was gradually released from NPs over 24h, which is sufficient time for targeting and treatment of the NPs in inflamed arthritis via intravenous injection. In vitro study using splenocytes excised from spleens of mice following induction of arthritis using collagen clearly demonstrated the anti-proliferative activity of TAC HSA-NPs on activated T cells compared with non-activated T cells. Furthermore, TAC HSA-NPs displayed significantly more anti-arthritic activity than TAC formulations including intravenously administered TAC solution or oral TAC suspension, as reflected by the incidence of arthritis and clinical score (1.6 vs. 3.2 and 5.0, respectively). These improvements were due to the targetability of HSA that facilitated the accumulation of TAC HSA-NPs at inflamed arthritis sites. TAC HSA-NPs are a promising drug delivery system to enhance water solubility and increase accumulation in joints for treatment of rheumatoid arthritis.

Facile one-pot formulation of TRAIL-embedded paclitaxel-bound albumin nanoparticles for the treatment of pancreatic cancer.

Nanoparticle albumin-bound (nab™) technology is an effective way of delivering hydrophobic chemotherapeutics. We developed a one-pot/one-step formulation of paclitaxel (PTX)-bound albumin nanoparticles with embedded tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/PTX HSA-NP) for the treatment of pancreatic cancer. TRAIL/PTX HSA-NPs were fabricated using a high-pressure homogenizer at a TRAIL feeding ratio of 0.2%, 1.0%, and 2.0%. TRAIL/PTX HSA-NPs were spherical and became larger in size (170-230 nm) with increasing TRAIL amount (0.2-2.0%). The loading efficiencies of PTX were in the range of ∼86.4% and significantly low at 2.0% TRAIL (60.4%). Specifically, the inhibitory concentrations (IC50) of TRAIL (1.0 or 2.0%)/PTX HSA-NPs were >20-fold lower than that of plain PTX-HSA NP (0.032±0.06, 0.022±0.005, and 0.96±0.15 ng/ml, respectively) in pancreatic Mia Paca-2 cells. Considering TRAIL loading, bioactivity, and particle size, TRAIL(1.0%)/PTX HSA-NPs were determined as the optimal candidate for further studies. TRAIL(1.0%)/PTX HSA-NPs displayed substantially greater apoptotic activity than plain PTX HSA-NP in both FACS and TUNEL analysis. The loaded PTX and TRAIL were gradually released from the TRAIL(1.0%)/PTX HSA-NPs until ∼24 h, which is considered to be a sufficient time for delivery to the tumor tissue. TRAIL(1.0%)/PTX HSA-NP displayed markedly more antitumor efficacy than plain PTX HSA-NP in Mia Paca-2 cell-xenografted mice in terms of tumor volume (size) and weight (213.9 mm(3) and 0.18 g vs. 1126.8 mm(3) and 0.80 g, respectively). These improved in vitro and in vivo performances were due to the combined synergistic effects of PTX and TRAIL. We believe that this TRAIL/PTX HSA-NP would have potential as a novel apoptosis-based anticancer agent.

In situ facile-forming PEG cross-linked albumin hydrogels loaded with an apoptotic TRAIL protein.

The key to making a practicable hydrogel for pharmaceutical or medical purposes is to endow it with relevant properties, i.e., facile fabrication, gelation time-controllability, and in situ injectability given a firm basis for safety/biocompatibility. Here, the authors describe an in situ gelling, injectable, albumin-cross-linked polyethylene glycol (PEG) hydrogel that was produced using a thiol-maleimide reaction. This hydrogel consists of two biocompatible components, namely, thiolated human serum albumin and 4-arm PEG20k-maleimide, and can be easily fabricated and gelled in situ within 60s by simply mixing its two components. In addition, the gelation time of this system is controllable in the range 15s to 5min. This hydrogel hardly interacted with an apoptotic TRAIL protein, ensuring suitable release profiles that maximize therapeutic efficacy. Specifically, tumors (volume: 278.8mm(3)) in Mia Paca-2 cell-xenografted BALB/c nu/nu mice treated with the TRAIL-loaded HSA-PEG hydrogel were markedly smaller than mice treated with the hydrogel prepared via an amine-N-hydroxysuccinimide reaction or non-treated mice (1275.5mm(3) and 1816.5mm(3), respectively). We believe that this hydrogel would be a new prototype of locally injectable sustained-release type anti-cancer agents, and furthermore offers practical convenience for a doctor and universal applicability for a variety of therapeutic proteins.

PEGylated apoptotic protein-loaded PLGA microspheres for cancer therapy.

The aim of the current study was to investigate the antitumor potential of poly (D,L-lactic-co-glycolic acid) microspheres (PLGA MSs) containing polyethylene glycol (PEG)-conjugated (PEGylated) tumor necrosis factor-related apoptosis-inducing ligand (PEG-TRAIL). PEG-TRAIL PLGA MSs were prepared by using a water-in-oil-in-water double-emulsion method, and the apoptotic activities of supernatants released from the PLGA MSs at days 1, 3, and 7 were examined. The antitumor effect caused by PEG-TRAIL PLGA MSs was evaluated in pancreatic Mia Paca-2 cell-xenografted mice. PEG-TRAIL PLGA MS was found to be spherical and 14.4±1.06 µm in size, and its encapsulation efficiency was significantly greater than that of TRAIL MS (85.7%±4.1% vs 43.3%±10.9%, respectively). The PLGA MS gradually released PEG-TRAIL for 14 days, and the released PEG-TRAIL was shown to have clear apoptotic activity in Mia Paca-2 cells, whereas TRAIL released after 1 day had a negligible activity. Finally, PEG-TRAIL PLGA MS displayed remarkably greater antitumor efficacy than blank or TRAIL PLGA MS in Mia Paca-2 cell-xenografted mice in terms of tumor volume and weight, apparently due to increased stability and well-retained apoptotic activity of PEG-TRAIL in PLGA MS. We believe that this PLGA MS system, combined with PEG-TRAIL, should be considered a promising candidate for treating pancreatic cancer.

Inhalable self-assembled albumin nanoparticles for treating drug-resistant lung cancer.

Direct pulmonary delivery of anti-cancer agents is viewed as an effective way of treating lung cancer. Here, we fabricated inhalable nanoparticles made of human serum albumin (HSA) conjugated with doxorubicin and octyl aldehyde and adsorbed with apoptotic TRAIL protein (TRAIL/Dox HSA-NP). The octyl aldehyde and doxorubicin endowed HSA with significant hydrophobicity that facilitated self-assembly. TRAIL/Dox HSA-NP was found to have excellent particle size (~340nm), morphology, dispersability, and aerosolization properties. TRAIL/Dox HSA-NP displayed synergistic cytotoxicity and apoptotic activity in H226 lung cancer cells vs. HSA-NP containing TRAIL or Dox alone. TRAIL/Dox HSA-NP was well deposited in the mouse lungs using an aerosolizer, and TRAIL and Dox-HSA were found to be gradually released over 3days. The anti-tumor efficacy of pulmonary administered TRAIL/Dox HSA-NP was evaluated in BALB/c nu/nu mice bearing H226 cell-induced metastatic tumors. It was found that the tumors of H226-implanted mice treated with TRAIL/Dox HSA-NP were remarkably smaller and lighter than those of mice treated with TRAIL or Dox HSA-NP alone (337.5±7.5; 678.2±51.5; and 598.9±24.8mg, respectively). Importantly, this improved anti-tumor efficacy was found to be due to the synergistic apoptotic effects of Dox and TRAIL. In the authors' opinion, TRAIL/Dox HSA-NP offers a potential inhalable anti-lung cancer drug delivery system. Furthermore, the synergism displayed by combined use of Dox and TRAIL could be used to markedly reduce doxorubicin doses and minimize its side effects.

Human serum albumin-TRAIL conjugate for the treatment of rheumatoid arthritis.

Albumin conjugation is viewed as an effective means of protracting short in vivo lifespans of proteins and targeting rheumatoid arthritis (RA). In this study, we present a human serum albumin (HSA) conjugate linked with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) via a bifunctional PEG derivative (HSA-TRAIL). Prepared HSA-TRAIL was found to have a larger molecular size (∼240 kDa, 15.4 nm) than TRAIL (∼66 kDa, 6.2 nm), and its bioactivity (apoptosis, cytotoxicity, and antiproliferation) was well preserved in Mia Paca-2 cells and mouse splenocytes. The enhanced therapeutic efficacy of HSA-TRAIL was demonstrated in collagen-induced arthritis (CIA) mice. The incidence and clinical scores, expressed as degree of erythema and swelling in HSA-TRAIL-treated mice, were remarkably lower than those of TRAIL-treated mice. The serum levels of pro-inflammatory cytokines IFN-γ, TNF-α, IL-1ß, and IL-2 in HSA-TRAIL-treated mice were significantly lower than those of TRAIL-treated mice. Furthermore, HSA-TRAIL accumulated in the hind paws of CIA mice, not in naïve TRAIL mice. Pharmacokinetic profiles of HSA-TRAIL were greatly improved in comparison to those of TRAIL (AUCinf: 844.1 ± 130.0 vs 36.0 ± 1.2 ng·h/mL; t1/2: 6.20 ± 0.72 vs 0.23 ± 0.01 h, respectively). The HSA-TRAIL conjugate, which presents clear advantages of targeting RA and long systemic circulation by HSA and unique anti-inflammatory efficacy by TRAIL, has potential as a novel treatment for rheumatoid arthritis.

Apoptotic activity and antitumor efficacy of PEGylated TNF-related apoptosis-inducing ligand (TRAIL) in a Mia Paca-2 cell-xenografted mouse model.

The purpose of this study was to demonstrate the apoptotic activity and antitumor effect of PEGylated tumor necrosis factor-related apoptosis-inducing ligand (PEG-TRAIL) in pancreatic carcinoma Mia Paca-2 cells and in Mia Paca-2 cell-xenografted mice. PEG-TRAIL was prepared using mPEG-aldehyde (Mw 5 kDa). The apoptosis induced by PEG-TRAIL in Mia Paca-2 cells and in the tumors of Mia Paca-2 cell-xenografted mice was quantified by FACS analysis and using a TUNEL assay. Mia Paca-2 cell-xenografted BALB/c nu/nu mice were administered intratumoral injections of PEG-TRAIL (50 µg/mouse/injection) every 3 days from day 0 (~4 weeks after xenografting) to day 15. Tumor volumes were measured every 3 days from day 0 to day 27. PEG-TRAIL displayed obvious apoptotic activity in Mia Paca-2 cells; the FACS signal was shifted to the apoptotic area and the cells exhibited green fluorescence indicating apoptosis in the TUNEL assay. Furthermore, PEG-TRAIL was found to suppress tumors in Mia Paca-2 cell-xenografted mice (tumor volumes: 183.9±134.1 for PEG-TRAIL vs. 1827.3±264.5 mm(3) for saline control). In addition, in vivo TUNEL assays of tumor tissues showed that the antitumor effect of PEG-TRAIL was due apoptosis. Our findings provide clear in vivo evidence of the antitumor potential of PEG-TRAIL in a Mia Paca-2 cell-xenografted mouse model based of pancreatic cancer.

Four-arm PEG cross-linked hyaluronic acid hydrogels containing PEGylated apoptotic TRAIL protein for treating pancreatic cancer.

Four-arm polyethylene glycol (PEG) cross-linked hyaluronic acid (HA) hydrogels containing PEGylated tumor necrosis factor-related apoptosis-inducing ligand (PEG-TRAIL) were fabricated, and their antitumor effects were evaluated in pancreatic cell (Mia Paca-2)-xenografted mice. HA was conjugated with 4-arm PEG(10k)-amine (a cross-linker) at ratios of 100:1 and 100:2 using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride as a cross-linker, and TRAIL or PEG-TRAIL was incorporated into these HA hydrogels. HA hydrogels at a 100:1 ratio were prepared in good yields (>88%), were moderately stiff, and gradually released PEG-TRAIL over ~14 days in vitro and over ~7 days in vivo (as determined by high-pressure liquid chromatography and infrared imaging). The released PEG-TRAIL was found to have obvious apoptotic activity in Mia Paca-2 cells. PEG-TRAIL HA hydrogels displayed remarkably more antitumor efficacy than TRAIL HA hydrogels in Mia Paca-2 cell-xenografted mice in terms of tumor volumes (size) and weights (453.2mm(3) and 1.03 g vs. 867.5mm(3) and 1.86 g). Furthermore, this improved antitumor efficacy was found to be due to the apoptotic activity of PEG-TRAIL in vivo (determined by a TUNEL assay) despite its substantially lower cytotoxicity than native TRAIL (IC50 values: 71.8 and 202.5 ng ml(-1), respectively). This overall enhanced antitumor effect of PEG-TRAIL HA hydrogels appeared to be due to the increased stability of PEGylated TRAIL in HA hydrogels. These findings indicate that this HA hydrogel system combined with PEG-TRAIL should be considered a potential candidate for the treatment of pancreatic cancer.

Decanoic acid-modified glycol chitosan hydrogels containing tightly adsorbed palmityl-acylated exendin-4 as a long-acting sustained-release anti-diabetic system.

Decanoic acid-modified glycol chitosan (DA-GC) hydrogels containing tightly adsorbed palmitic acid-modified exendin-4 (Ex4-C16) were prepared, and their pharmaceutical abilities as a long-acting sustained-release exendin-4 system for the treatment of diabetes were evaluated. Glycol chitosan (GC) was conjugated with N-hydroxysuccinimide-activated decanoic acid (DA) in anhydrous 0.4% dimethylaminopyridine/dimethylsulfoxide at different feed ratios. DA-GC hydrogels formed by physical self-assembly during dialysis vs. deionized water, and their inner network structures, swelling or gel-forming abilities and release properties were examined. The hypoglycemia caused by Ex4-C16-loaded DA-GC hydrogels was evaluated by subcutaneous administration in type 2 diabetic db/db mice. The results obtained showed that GC prepared at a DA:GC feed ratio of 1:100 had optimal properties with respect to hydrogel swelling, stiffness and Ex4-C16 incorporation, whereas DA-GC hydrogels prepared at a feed ratio of greater than 1:100 formed gels that were too stiff. The in vitro and in vivo release of Ex4-C16 from DA-GC hydrogels was dramatically delayed compared with native Ex4 probably due to strong hydrophobic interactions. In particular, Ex4-C16 in DA-GC hydrogels was found to be present around the injection site up to 10 days after subcutaneous administration, whereas Ex4 in DA-GC hydrogels was cleared from injection sites in ≈ 2 days in ICR mice. Finally, the hypoglycemia induced by Ex4-C16 DA-GC hydrogels was maintained for >7 days. Our findings demonstrate that Ex4-C16 DA-GC hydrogels offer a potential delivery system for the long-term treatment of type 2 diabetes.

Doxorubicin-loaded porous PLGA microparticles with surface attached TRAIL for the inhalation treatment of metastatic lung cancer.

Inhalable highly porous large PLGA microparticles with incorporated doxorubicin and surface-attached with TRAIL (TRAIL/Dox PLGA MP) were fabricated using a w/o/w double emulsification method using ammonium bicarbonate as a gas-foaming agent for the treatment of lung cancer. The TRAIL/Dox PLGA MP produced were highly porous and 11.5 ± 0.4 µm in diameter, and the loading efficiencies of Dox and TRAIL were 86.5 ± 6.5% and 91.8 ± 2.4%, respectively. TRAIL and doxorubicin were gradually released by TRAIL/Dox PLGA over 7 days, and pulmonary administration resulted in the deposition of TRAIL/Dox PLGA MP in mouse lungs, and they remained in situ for up to a week. The anti-tumor efficacy of pulmonary administered TRAIL/Dox PLGA MP was evaluated in a BALB/c nu/nu mice mouse model of H226 cell metastasis. Tumors in H226-implanted mice treated with TRAIL/Dox PLGA MP were markedly smaller and fewer in number than mice treated with TRAIL or Dox PLGA MP alone. Furthermore, this improved performance was found to be due to the synergistic apoptotic effects of the two drugs. We believe that TRAIL/Dox PLGA MP offer a promise of a sustained-release, long-acting, inhalable anti-lung cancer agent. Furthermore, the synergism observed between TRAIL and doxorubicin suggests that the doxorubicin dosage could be substantially reduced and its side effects minimized.

Doxorubicin-loaded highly porous large PLGA microparticles as a sustained- release inhalation system for the treatment of metastatic lung cancer.

Doxorubicin-loaded highly porous large PLGA microparticles (Dox PLGA MPs) were prepared using a w/o/w double emulsification method using ammonium bicarbonate effervescent salt. The prepared Dox PLGA MPs were characterized by particle size analysis, scanning electron microscopy, and confocal microscopy. In vitro cytotoxicity to B16F10 melanoma cells and lung deposition in C57BL/6 mice were examined, and finally the anti-tumor efficacy of pulmonary administered Dox PLGA MPs was evaluated in a mouse model of B16F10 melanoma metastasis. Results showed that Dox PLGA MPs were highly porous, had high encapsulation efficiency, and good aerosolization characteristics. Doxorubicin was gradually released from Dox PLGA MPs over 2 weeks, and after pulmonary administration, Dox PLGA MPs were deposited in lungs and remained in situ for up to 14 days. Furthermore, exposure to Dox PLGA MPs killed B16F10 cells in vitro within 24 h. In particular, tumors in B16F10-implanted mice treated with Dox PLGA MPs were remarkably smaller in terms of mass and number than those in non-treated B16F10-implanted mice. We believe that doxorubicin-loaded highly porous large PLGA microparticles have great potential as a long-term inhalation agent for the treatment of lung cancer.