Novel Self-Assembled Micelles With Increased Tumor Penetration and Anti-Tumor Efficiency Against Breast Cancer.

PURPOSE: Recently, docetaxel (DTX) micelles based on retinoic acid derivative surfactants showed lower systemic toxicity and bioequivalence to polysorbate-solubilized docetaxel (Taxotere®) in a phase II clinical study. However, the poor stability of these surfactants in vitro and in vivo led to extremely harsh storage conditions with methanol, and the formed micelles were quickly disintegrated with rapid drug burst release in vivo. To further enhance the stability and accumulation in tumors of DTX micelles, a novel surfactant based on acitretin (ACMeNa) was synthesized and used to prepare DTX micelles to improve anti-tumor efficiency. METHODS: Novel micelle-forming excipients were synthesized, and the micelles were prepared using the thin film hydration technique. The targeting effect in vitro, distribution in the tumor, and its mechanism were observed. Pharmacokinetics and anti-tumor effect were further investigated in rats and tumor-bearing female mice, respectively. RESULTS: The DTX-micelles prepared with ACMeNa (ACM-DTX) exhibited a small size (21.9 ± 0.3 nm), 39% load efficiency, and excellent stability in vitro and in vivo. Long circulation time, sustained and steady accumulation, and strong penetration in the tumor were observed in vivo, contributing to a better anti-tumor effect and lower adverse effects. CONCLUSIONS: The micelles formed by ACMeNa showed a better balance between anti-tumor and adverse effects. It is a promising system for delivering hydrophobic molecules for cancer therapy.

Discovery of a Long-Acting Parathyroid Hormone 1-34 Analogue to Treat Hypoparathyroidism.

Hypoparathyroidism (HP) is a rare disease with clinical manifestations of hypocalcemia and hyperphosphatemia, resulting from deficient or absent parathyroid hormone (PTH) secretion. Conventional treatment for patients with HP involves extensive calcium and vitamin D supplementation. In 2015, PTH1-84 was approved by the United States Food and Drug Administration as an adjunct for HP patients who cannot be well-controlled on conventional treatment. However, PTH1-84 therapy requires a daily injection, leading to poor patient compliance. The purpose of this study was to develop a long-acting PTH1-34 analogue by increasing its affinity to albumin. Three PTH1-34 variants were generated by substituting two of the three lysine (Lys) residues with arginine, reserving a single Lys as the modification site in each sequence. A series of side chains, containing fatty acid, deoxycholic acid, or biotin groups, were synthesized to modify these PTH1-34 variants by using a solid-liquid phase synthesis approach. In vitro bioactivity and albumin affinity tests were used to screen these new PTH1-34 analogues. Finally, Lys27-AAPC was selected from 69 synthesized analogues as a candidate therapeutic compound because it retained potency and exhibited a high albumin-binding capacity. In pharmacodynamic experiments, Lys27-AAPC demonstrated enhanced and prolonged efficacy in serum calcium elevating relative to PTH1-84. Moreover, a lyophilized powder for injection containing Lys27-AAPC was developed for further testing and represented a potential long-acting HP treatment.

Cardioprotective activity of placental growth factor in a rat model of acute myocardial infarction: nanoparticle-based delivery versus direct myocardial injection.

BACKGROUND: To comparatively evaluate the cardioprotective activity of placental growth factor (PGF) delivered through direct injection and a nanoparticle-based system respectively and to study the underlying mechanisms in a rat model of acute myocardial infarction (AMI). METHODS: Poly lactic-co-glycolic acid (PLGA)-based PGF-carrying nanoparticles (PGF-PLGANPs) were created. The mean size and morphology of particles were analyzed with particle size analyzer and transmission electronic microscopy (TEM). Encapsulation efficiency and sustained-release dose curve were analyzed by ELISA. Sprague-Dawley rats were randomized into four groups (n = 10). While animals in the first group were left untreated as controls, those in the other 3 groups underwent surgical induction of AMI, followed by treatment with physiological saline, PGF, and PGF-PLGANPs, respectively. Cardiac function was evaluated by transthoracic echocardiography at 4 weeks after treatment. At 6 weeks, rats were sacrificed, infarction size was analyzed with Masson trichrome staining, and protein contents of TIMP-2, MT1-MMP and MMP-2 at the infarction border were determined by immunohistochemistry and western blotting analysis. RESULTS: PGF was released for at least 15 days, showing successful preparation of PGF-PLGANPs. Coronary artery ligation successfully induced AMI. Compared to physiological saline control, PGF, injected to the myocardium either as a nude molecule or in a form of nanoparticles, significantly reduced infarction size, improved cardiac function, and elevated myocardial expression of TIMP-2, MT1-MMP, and MMP-2 (P < 0.05). The effect of PGF-PLGANPs was more pronounced than that of non-encapsulated PGF (P < 0.05). CONCLUSION: Target PGF delivery to myocardium may improve cardiac function after AMI in rats. PLGA-based nanoparticles appear to be a better approach to delivery PGF. PGF exerts its cardioprotective effect at least partially through regulating metalloproteinase-mediated myocardial tissue remodeling.

Structure-property relationship in the evaluation of xanthan gum functionality for oral suspensions and tablets.

The functional properties of xanthan gum (XG) in pharmaceutical preparations depend on its rheological properties, which inevitably rely on its molecular structure. Hence, this work investigated the relationship between the molecular structure of XG and its rheological properties and functional characteristics, and revealed the structural factors influencing the XG functionalities in oral suspensions and matrix tablets. Primarily, the molecular structures of four commercial XG products were characterized by infrared spectroscopy, differential scanning calorimetry and measuring the monosaccharide composition, average molecular weight, and pyruvate and acetyl contents. Furthermore, the flow behavior and viscoelasticity of XG solutions, the viscoelasticity of XG hydrogels, and XG combinations (XGC, aqueous solution containing XG, liquid glucose, and glycerin) were investigated. Finally, the dissolution time of XGC and the swelling and erosion properties of the XG matrix were studied to evaluate XG functionality in oral suspensions and matrix tablets, respectively. Results showed that the polydispersity of molecular weight and the pyruvate content affected the functionality and performance of XG in suspension and tablet forms. The higher polydispersity and pyruvate content of XG improved the hydrogel strength, which led to a longer dissolution time of XGC and a higher swelling extent of the XG matrix but a slower erosion rate.

Selective ischemic-hemisphere targeting Ginkgolide B liposomes with improved solubility and therapeutic efficacy for cerebral ischemia-reperfusion injury.

Cerebral ischemia-reperfusion injury (CI/RI) remains the main cause of disability and death in stroke patients due to lack of effective therapeutic strategies. One of the main issues related to CI/RI treatment is the presence of the blood-brain barrier (BBB), which affects the intracerebral delivery of drugs. Ginkgolide B (GB), a major bioactive component in commercially available products of Ginkgo biloba, has been shown significance in CI/RI treatment by regulating inflammatory pathways, oxidative damage, and metabolic disturbance, and seems to be a candidate for stroke recovery. However, limited by its poor hydrophilicity and lipophilicity, the development of GB preparations with good solubility, stability, and the ability to cross the BBB remains a challenge. Herein, we propose a combinatorial strategy by conjugating GB with highly lipophilic docosahexaenoic acid (DHA) to obtain a covalent complex GB-DHA, which can not only enhance the pharmacological effect of GB, but can also be encapsulated in liposomes stably. The amount of finally constructed Lipo@GB-DHA targeting to ischemic hemisphere was validated 2.2 times that of free solution in middle cerebral artery occlusion (MCAO) rats. Compared to the marketed ginkgolide injection, Lipo@GB-DHA significantly reduced infarct volume with better neurobehavioral recovery in MCAO rats after being intravenously administered both at 2 h and 6 h post-reperfusion. Low levels of reactive oxygen species (ROS) and high neuron survival in vitro was maintained via Lipo@GB-DHA treatment, while microglia in the ischemic brain were polarized from the pro-inflammatory M1 phenotype to the tissue-repairing M2 phenotype, which modulate neuroinflammatory and angiogenesis. In addition, Lipo@GB-DHA inhibited neuronal apoptosis via regulating the apoptotic pathway and maintained homeostasis by activating the autophagy pathway. Thus, transforming GB into a lipophilic complex and loading it into liposomes provides a promising nanomedicine strategy with excellent CI/RI therapeutic efficacy and industrialization prospects.

Multifunctional ROS-Responsive and TME-Modulated Lipid-Polymer Hybrid Nanoparticles for Enhanced Tumor Penetration.

Purpose: To enhance tumor penetration by formulation design and tumor microenvironment (TME) modulation, herein a novel reactive oxygen species (ROS)-responsive size/shape transformable lipid-polymer hybrid nanoparticle (LPN) has been fabricated for the co-delivery of an anticancer and collagen-inhibition drug. Methods: A ROS-responsive poly(D, L-lactide)-thioketal-polyethylene glycol (PLA-TK-PEG) co-polymer was synthesized. LPNs were then fabricated by encapsulation of losartan (LST)-loaded micelles as the core to support paclitaxel (PTX)-loaded liposomes. The PEG content in the lipid shell of LPNs was then adjusted to obtain the size-/shape-transformable LPNs (M/LST-Lip/PTX-PEG5%). The ROS-responsiveness was observed in vitro by transmission electron microscopy and the tumor-penetration of the LPNs was evaluated in 3D tumor spheroids by confocal laser scanning microscopy. Tumor-targeting, tumor-penetrating, and antitumor efficacies of the NPs in 4T1 tumor-bearing mice were determined by in vivo imaging. Results: ROS-responsive micellar core degradation and the transformation of spherical LPNs (120nm) to smaller 40 mm discoid nanoparticles (NP) were observed. The transformable LPNs exhibited enhanced capacity of penetration in contrast to the un-transformable preparations in three-dimensional (3D) tumor spheroids. Furthermore, synergetic penetrating enhancement was achieved by LST-loaded transformable LPNs in 4T1 and fibroblast cell mixed 3D tumor spheroids. The improved tumor penetration of LST-loaded transformable LPNs was observed in vivo, which could be due to their collagen inhibiting and size/shape transformable effect. Due to their enhanced penetrability, LST and PTX-loaded transformable LPNs demonstrated significant in vivo antitumor efficacy in comparison to other preparations. Conclusion: The results confirmed the efficacy of M/LST-Lip/PTX-PEG5% in tumor targeting, collagen inhibition in TME, and enhanced tumor penetration. This novel drug delivery system can therefore play a substantial role in improving the therapeutic efficacy of antitumor drugs combined with TME-improving agents.

Development of a stable single-vial liposomal formulation for vincristine.

Background: Vincristine is a potent therapeutic agent with well-defined activity against hematologic malignancies and solid tumors. It is a cell-cycle specific drug with concentration and exposure duration dependent activity. When used by liposomal delivery, it exhibits enhanced anti-tumor activity. However, vincristine liposome formulation in the clinic is supplied as a 3-vial-kit due to lacking sufficient stability. So it has to be prepared in situ prior to use through a multi-step process. Purpose: The purpose here is to develop a more stable and ready-to-use liposomal formulation for vincritstine in one vial. Patients and methods: A series of preparations were investigated based on sphingomyelin/cholesterol/PEG2000-DSPE lipid composition, with different drug/lipid (D/L) ratios (1/10, 1/5, 1/2), using an active sucrose octasulfate triethylamine salt gradient loading method. In this work, compared to generic vincristine sulfate liposome injection (GVM), the stability both in vivo and in vitro and efficacy in vivo of novel vincristine liposomes were investigated. Results: It was shown that the degradation of vincristine during 2-8°C storage was significantly decreased from 8.2% in 1 month (GVM) to 2.9% in 12 months (D/L ratio 1/5). The half-time for sphingomyelin/cholesterol/PEG2000-DSPE liposomes in vivo could be adjusted from 17.4 h (D/L ratio 1/10) to 22.7 h (D/L ratio 1/2) in rats, while the half-time for GVM was only 11.1 h. The increase in drug retention contributed to the lower in vivo toxicity. The antitumor efficacy was evaluated using a human melanoma tumor model and showed remarkable improvement compared to GVM. Conclusion: The study demonstrates that the new formulation with the drug/lipid ratio of 1/5 owns a higher encapsulation efficiency, better stability, lower toxicity and superior antitumor efficacy, which is screened out for further development.

Menthol-modified casein nanoparticles loading 10-hydroxycamptothecin for glioma targeting therapy.

Chemotherapy outcomes for the treatment of glioma remains unsatisfactory due to the inefficient drug transport across the blood-brain barrier (BBB) and insufficient drug accumulation in the tumor region. Although many approaches, including various nanosystems, have been developed to promote the distribution of chemotherapeutics in the brain tumor, the delivery efficiency and the possible damage to the normal brain function still greatly restrict the clinical application of the nanocarriers. Therefore, it is urgent and necessary to discover more safe and effective BBB penetration and glioma-targeting strategies. In the present study, menthol, one of the strongest BBB penetration enhancers screened from traditional Chinese medicine, was conjugated to casein, a natural food protein with brain targeting capability. Then the conjugate self-assembled into the nanoparticles to load anti-cancer drugs. The nanoparticles were characterized to have appropriate size, spheroid shape and high loading drug capacity. Tumor spheroid penetration experiments demonstrated that penetration ability of menthol-modified casein nanoparticles (M-CA-NP) into the tumor were much deeper than that of unmodified nanoparticles. In vivo imaging further verified that M-CA-NPs exhibited higher brain tumor distribution than unmodified nanoparticles. The median survival time of glioma-bearing mice treated with HCPT-M-CA-NPs was significantly prolonged than those treated with free HCPT or HCPT-CA-NPs. HE staining of the organs indicated the safety of the nanoparticles. Therefore, the study combined the advantages of traditional Chinese medicine strategy with modern delivery technology for brain targeting, and provide a safe and effective approach for glioma therapy.

Microenvironment Remodeling Micelles for Alzheimer's Disease Therapy by Early Modulation of Activated Microglia.

Current strategies for Alzheimer's disease (AD) treatments focus on pathologies in the late stage of the disease progression. Poor clinical outcomes are displayed due to the irreversible damages caused by early microglia abnormality which triggers disease development before identical symptoms emerge. Based on the crosstalk between microglia and brain microenvironment, a reactive oxygen species (ROS)-responsive polymeric micelle system (Ab-PEG-LysB/curcumin (APLB/CUR)) is reported to normalize the oxidative and inflammatory microenvironment and reeducate microglia from an early phase of AD. Through an ß-amyloid (Aß) transportation-mimicked pathway, the micelles can accumulate into the diseased regions and exert synergistic effects of polymer-based ROS scavenging and cargo-based Aß inhibition upon microenvironment stimuli. This multitarget strategy exhibits gradual correction of the brain microenvironment, efficient neuroprotection, and microglia modulation, leading to decreased Aß plaque burdens and consequently enhanced cognitive functions in APPswe/PSEN1dE9 model mice. The results indicate that microglia can be exploited as an early target for AD treatment and their states can be controlled via microenvironment modulation.

Microthrombus-Targeting Micelles for Neurovascular Remodeling and Enhanced Microcirculatory Perfusion in Acute Ischemic Stroke.

Reperfusion injury exists as the major obstacle to full recovery of neuron functions after ischemic stroke onset and clinical thrombolytic therapies. Complex cellular cascades including oxidative stress, neuroinflammation, and brain vascular impairment occur within neurovascular units, leading to microthrombus formation and ultimate neuron death. In this work, a multitarget micelle system is developed to simultaneously modulate various cell types involved in these events. Briefly, rapamycin is encapsulated in self-assembled micelles that are consisted of reactive oxygen species (ROS)-responsive and fibrin-binding polymers to achieve micelle retention and controlled drug release within the ischemic lesion. Neuron survival is reinforced by the combination of micelle facilitated ROS elimination and antistress signaling pathway interference under ischemia conditions. In vivo results demonstrate an overall remodeling of neurovascular unit through micelle polarized M2 microglia repair and blood-brain barrier preservation, leading to enhanced neuroprotection and blood perfusion. This strategy gives a proof of concept that neurovascular units can serve as an integrated target for ischemic stroke treatment with nanomedicines.

Preparation and characterization of intravenously injectable nimodipine nanosuspension.

The purpose of this study was to develop an alternative, improved and better tolerated injectable nimodipine nanosuspension compared with commercially available ethanol solution. In this study, nimodipine nanosuspension was prepared by high-pressure homogenization (HPH). The effects of the production parameters such as pressure, cycle numbers and crushing principles on the mean particle size, 99% diameter and polydispersity of the nanosuspension were investigated. Characterization of the product was performed by scanning electron microscope (SEM) and differential scanning calorimeter (DSC). The safety of the nimodipine nanosuspension was discussed with special attention to contamination by microparticles and the increase in saturation solubility C(s). Irritability study in rabbits showed that this formulation provided less local irritation and phlebitis risks than the commercial ethanol product, which represented a promising new drug formulation for intravenous therapy of subarachnoid hemorrhage (SAH)-related vasospasm.

Distribution of an intravenous injectable nimodipine nanosuspension in mice.

The distribution of an intravenous injectable nimodipine nanosuspension with mean particle size of both 300 and 650 nm in mice was systemically investigated compared with that of a nimodipine ethanol formulation (Nimotop) and a nanosuspension coated with Tween-80. The results showed that the 650-nm nanoparticles provided significantly higher drug concentrations in the liver, spleen and lungs because of their capture by Kupffer cells in the mononuclear phagocyte system, but lower drug concentrations in the brain compared with Nimotop and smaller nanoparticles. These nanoparticles failed to give increased brain concentrations even when coated with Tween-80. The 300-nm nanoparticles could effectively increase drug concentrations in the brain and remarkably reduce drug concentrations in the liver, spleen and lungs, indicating that the nimodipine nanosuspension may be a promising formulation with no ethanol, but the particle size must be small.

Preparation, characterization, and evaluation of liposomal ferulic acid in vitro and in vivo.

In the present study, various gradients were evaluated for efficient loading of weak acid into liposomes. Several salt gradients showed efficient loading of ferulic acid (FA) into liposomes and the optimized conditions were established in calcium acetate gradient method to obtain 80.2 +/- 5.2% entrapment efficiency (EE). Unilamellar vesicles were observed in micrographs and liposomal FA showed good stability. 80% of FA was released from liposomes within 5 h in vitro. There is a novel finding in this study: that drugs could be entrapped with a high solubility in the intraliposomal buffer in contrast to the low solubility in the extraliposomal buffer. The results of body distribution in rats indicated that liposomes could improve the body distribution of FA. For FA liposome, the concentration of FA in brain was two-fold higher than that of free FA. Liposomal FA was a promising approach to improve the body distribution of FA.

Natural Brain Penetration Enhancer-Modified Albumin Nanoparticles for Glioma Targeting Delivery.

The unsatisfactory therapeutic outcome for glioma is mainly due to the poor blood-brain barrier (BBB) permeability and inefficient accumulation in the glioma area of chemotherapeutic agents. The existing drug delivery strategies can increase drug transport to the brain but are restricted by side effects and/or poor delivery efficiency. In this study, potent brain penetration enhancers were screened from the active components of aromatic resuscitation drugs used in traditional Chinese medicine. A novel glioma-targeting system based on enhancer-modified albumin nanoparticles was developed to safely and efficiently deliver drugs to the glioma regions in the brain. The nanoparticles improved the transport of nanoparticles across brain capillary endothelial cell (BCEC) monolayer by increasing endocytosis in endothelial cells and causing BBB disruption. In vivo imaging studies demonstrated that the systems could enter the brain and subsequently accumulate in glioma cells with a much higher targeting efficiency than that of transferrin-modified albumin nanoparticles. Of note, the nanoparticles could be captured and penetrate through endothelial cells fenestrae in pineal gland, which is suggestive of an effective way to deliver a nanosystem to the brain by bypassing the BBB. The nanoparticles showed good biocompatibility and negligible cytotoxicity. The results reveal an efficient and safe strategy for brain drug delivery in glioma therapy.

[Freeze-drying of silymarin-loaded solid lipid nanoparticles (SM-SLN)].

OBJECTIVE: To investigate lyophilization of SM-SLN. METHOD: The parameters of lyophilization process was optimized. In addition, the protective effect of various types and concentrations of cryoprotectants were tested by shape, colour and disparity. RESULT: The mixture of 2% lactose and 2% glucose could better prevent nanoparticles from aggregating, the optimal lyophilization process was followed: precooled at -45 degrees C for 10 hr; primary drying at -25 degrees C for 5 hr; secondary drying at 10 degrees C for 3 hr; finally drying at 30 degrees C for 6 hr. CONCLUSION: Changes in particle size distribution during lyophilization could be minimized by optimizing the parameters of the lyophilization process and adding supporting agent.