In Vitro Drug Loading, Releasing Profiles, and In Vivo Embolic Efficacy and Safety Evaluation of a Novel Drug-Eluting Microsphere (CalliSpheres).

Background: To investigate morphology, physical property, loadability, stability, and release profiles of a novel drug-eluting microsphere, CalliSpheres, in vitro and to explore its embolic efficacy and safety in vivo. Materials and Methods: CalliSpheres (50-150 µm, 100-300 µm, and 300-500 µm) and doxorubicin in different amounts (20, 40, 80, and 100 mg) and concentrations (5 and 10 mg/mL) were prepared for experiments. Dynamic light scattering and an Agilent 1260 high-performance liquid chromatography system were used to quantify bead diameters and the efficiency of drug loading and release, respectively. Twelve New Zealand rabbits were treated with catheter-aided hepatic embolization using CalliSpheres. Results: CalliSpheres displayed a red color after loading with doxorubicin, and the mean diameters decreased by 20.7-25.8%. Almost 100% of the drug was incorporated with CalliSpheres in different sizes immersed with doxorubicin 20 mg, while loading efficiency ranged from 75.8% to 100.0% with doxorubicin at 40, 80, and 100 mg dependent on CalliSpheres sizes (smaller sizes, higher loading efficiency). Elevated loading efficiency was observed at higher concentration of doxorubicin solutions. Regarding release profiles, doxorubicin was released from CalliSpheres quickly at the very beginning, and doxorubicin release percentage was increased in the 50-150 µm group (39.2% ± 1.2%) compared with the 100-300 µm group (31.3% ± 1.3%) and 300-500 µm group (31.7% ± 2.5%). Digital subtraction angiography, computed tomography, and histopathologic emanation results proved in vivo safety and embolic efficacy of CalliSpheres. Conclusions: CalliSpheres present with good physical characteristics and satisfactory loading and releasing profiles in vitro and are well tolerated and efficient in embolization in vivo.

Assessment of Irinotecan Loading and Releasing Profiles of a Novel Drug-Eluting Microsphere (CalliSpheres) In Vitro.

Background: This study investigated irinotecan loading efficiency and release profiles of CalliSpheres in vitro. Materials and Methods: CalliSpheres with size of 50-150, 100-300, and 300-500 µm and irinotecan at different amounts (20, 40, 80, and 100 mg) and concentrations (5 and 10 mg/mL) were prepared for experiments. Dynamic light scattering and Agilent 1260 high-performance liquid chromatography system were used to quantify bead diameters and the efficiency of irinotecan loading and releasing properties, respectively. Results: The diameters of CalliSpheres with all sizes were reduced after being loaded with irinotecan compared with unloaded ones with shrinkage rate ranging from 8.5% to 16.2%. Above 80% irinotecan was incorporated with CalliSpheres with all sizes when being loaded with irinotecan 20, 40, and 80 mg, while loading efficiencies were 70%-80% when being loaded with irinotecan 100 mg. Besides, elevated loading efficiency was observed at a higher concentration of irinotecan solutions (10 mg/mL) compared with a lower concentration (5 mg/mL) for CalliSpheres with all sizes. As to release profiles, irinotecan was released from CalliSpheres very quickly, and irinotecan release rate was elevated in CalliSpheres with smaller size than CalliSpheres with larger size within the first 12 h, whereas it was similar among CalliSpheres with different sizes at 24 and 48 h with maximum release rate ∼100%. In addition, fetal bovine serum seemed to have an effect on the accelerating irinotecan release. Conclusion: CalliSpheres exhibits good physical characteristics, satisfied irinotecan loading efficiency, and acceptable releasing profiles.

Click-Chemistry Approach toward Antibacterial and Degradable Hybrid Hydrogels Based on Octa-Betaine Ester Polyhedral Oligomeric Silsesquioxane.

An efficient process for the synthesis of degradable hydrogels containing octa-betaine ester polyhedral oligomeric silsesquioxane (POSS) through efficient thiol-ene and Menschutkin click reactions was developed. The hydrogels exhibited a yield strength of 0.36 MPa and a compressive modulus of 4.38 MPa and displayed excellent flexibility as well as torsion resistance. Antibacterial efficacy of hydrogels (and degradation products) was evaluated using Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive). Efficacy was found to increase with the concentration of cetyl chloroacetate (CCA) in the hydrogel network, reaching 93% and 99% for Escherichia coli and Staphylococcus aureus, respectively. Degradation of hydrogels was observed in weak alkali conditions (pH = 8) and at physiological conditions (pH = 7.4). The degradation time of the hydrogels could be finely tuned by variation of the CCA content in the hydrogel and environmental stimulus. The tunable degradation behavior under physiological conditions combined with high antibacterial efficacy could render the presented materials interesting for tissue engineering applications.

Pulmonary-Affinity Paclitaxel Polymer Micelles in Response to Biological Functions of Ambroxol Enhance Therapeutic Effect on Lung Cancer.

PURPOSE: Cancer chemotherapy effect has been largely limited by cell autophagy and little drug accumulation at the action sites. Herein, we designed an intelligent strategy involving paclitaxel (PTX) polymer micelles in response to biological functions of ambroxol (Ax). The amphiphilic polymers polyethyleneglycol-polylactic acid (PEG-PLA) and Pluronic P105 were selected as nanocarriers to encapsulate PTX to form into lung affinity PEG-PLA/P105/PTX micelles. Ax which can up-regulate the secretion of pulmonary surfactant (PS) and inhibit autophagy was hired to change the microenvironment of the lung, thereby promoting the lung accumulation and increasing cell-killing sensitivity of the micelles. METHODS: The physical and chemical properties of the micelles were characterized including size, morphology, critical micellar concentration (CMC) and in vitro drug release behavior. The therapeutic effects of the combination regimen were characterized both in vitro and in vivo including study on Ax in promoting the secretion of pulmonary surfactant, in vitro cytotoxicity, cellular uptake, Western blotting, in vivo biodistribution, in vivo pharmacokinetics and in vivo antitumor efficacy. RESULTS: The PEG-PLA/P105/PTX micelles showed a particle size of 16.7 ± 0.5 nm, a nearly round shape, small CMC and sustained drug release property. Moreover, the in vitro results indicated that Ax could increase PS and LC3 protein secretion and enhance the cytotoxicity of PEG-PLA/P105/PTX micelles toward A549 cells. The in vivo results indicated that the combination therapeutic regimen could promote the micelles to distribute in lung and enhance the therapeutic effect on lung cancer. CONCLUSION: This multifunctional approach of modulating the tumor microenvironment to enhance drug transportation and cell-killing sensitivity in the action sites might offer a new avenue for effective lung cancer treatment.

A carbohydrate mimetic peptide modified size-shrinkable micelle nanocluster for anti-tumor targeting and penetrating drug delivery.

PURPOSE: To deliver the chemotherapeutics through the nanoparticles, the delivery system should accumulate at the tumor site first and then penetrate through the interstitium into the interior. The specific tumor-targeting pathway mediated via the receptor-ligand binding could achieve the desirable accumulation of nanoparticles, and the nanoparticles with smaller sizes were required for penetration. METHODS AND MATERIALS: We constructed a size-shrinkable nanocluster modified with a tumor-targeting motif IF-7 (IF-7-MNC) based on a pH-sensitive framework which could be disintegrated in an acid environment to release the micelles aggregated inside. The micelles were constructed by amphiphilic block copolymers PEG-PLA to encapsulate paclitaxel (PTX), while the cross-linked framework consisting of TPGS-PEI was used as a net to gather and release micelles. This nanoplatform could specifically bind with the tumor receptor Annexin A1 through the ligand IF-7 and then shrunk into small micelles with a desirable size for penetration. CONCLUSION: IF-7-MNC of 112.27±6.81 nm could shrink into micelles in PBS (0.01 M, pH 5.0) with sizes of 14.89±0.32 nm. The cellular-uptake results showed that IF-7-MNC could be significantly internalized by A549 cells and HUVEC cells, while the penetration of IF-7-MNC could be more prominent into the 3D-tumor spheroids compared with that of MNC. The biodistribution results displayed that the fluorescence of IF-7-MNC in the tumor site at 24 hrs was 4.5-fold stronger than that of MNC. The results of anti-tumor growth demonstrated that IF-7-MNC was more favorable for the tumor therapy than MNC, where the inhibitory rate of tumor growth was 88.29% in the PTX-loaded IF-7-MNC (IF-7-PMNC) treated group, significantly greater than PMNC treatment group (p<0.05).

Morphology, Loadability, and Releasing Profiles of CalliSpheres Microspheres in Delivering Oxaliplatin: An In Vitro Study.

OBJECTIVES: This study aimed to explore the morphology, loadability, and releasing profiles of CalliSpheres microspheres in delivering oxaliplatin. METHODS: Varied amount (20, 40, 60, and 80 mg oxaliplatin) and concentration (1.25, 2.5, 5.0 mg/mL oxaliplatin) of oxaliplatin were mixed with CalliSpheres microspheres with 3 sizes (50-150 µm, 100-300 µm, and 300-500 µm) to measure the loadability. Of all, 20 mg oxaliplatin-loaded CalliSpheres microspheres with 3 sizes was prepared to measure the releasing profiles, meanwhile, fetal bovine serum was added to determine the effect of serum on oxaliplatin releasing. The morphology and size distribution of CalliSpheres microspheres with 3 sizes before and after 20 mg oxaliplatin loading were detected. RESULTS: Oxaliplatin amount was negatively correlated with loading efficiency with highest loadability in 20 mg oxaliplatin group (maximum 40% in 50-100 µm CalliSpheres microspheres, 52% in 100-300 µm CalliSpheres microspheres, and 52% in 300-500 µm CalliSpheres microspheres), while oxaliplatin concentration was positively associated with loading efficiency. Similar drug-releasing profiles were observed among oxaliplatin-loaded CalliSpheres microspheres with 3 sizes, and a rapid drug release was discovered in CalliSpheres microspheres with 3 sizes as well. We also found that fetal bovine serum did not affect the drug-releasing profiles of oxaliplatin-loaded CalliSpheres microspheres. In addition, CalliSpheres microspheres was modified a little to ellipse shape and less smooth after oxaliplatin loading, and it was enlarged to some extent. CONCLUSION: This study discloses drug loadability, releasing profiles, and morphology change of CalliSpheres microspheres for delivering oxaliplatin, which provides potential evidences for application of oxaliplatin-loaded drug-eluting beads in clinical practice.

Leukocyte-mimicking Pluronic-lipid nanovesicle hybrids inhibit the growth and metastasis of breast cancer.

Breast cancer is a severe threat to the health of women, and the metastasis of tumor cells leads to high mortality in female patients. Evidence shows that leukocytes are recruited by breast tumors through adhesion to inflammatory endothelial cells as well as tumor cells. Moreover, it is known that Pluronic P123 is effective in the reduction of matrix metalloproteinases (MMPs), which play a key role in the degradation of the extracellular matrix (ECM), therefore helping tumor cells to escape from the primary site. Inspired by these mechanisms, we established a leukocyte-mimicking Pluronic-lipid nanovesicle hybrid (LPL) through integrating the membrane proteins extracted from leukocytes with membrane-like vesicles, with Pluronic P123 hybridized in the lipid bilayer, while paclitaxel (PTX) was selected as the model drug. The hybrid vesicles were perfectly incorporated with the leukocyte membrane proteins, and no disruption to the lipid membrane was caused by P123, with the bio-targeting ability of leukocytes and the MMP-9-downregulation effect of P123 fully preserved in LPL. LPL exhibited enhanced cellular uptake and anti-metastasis efficacy in in vitro assays, while significant tumor targeting capabilities were also found through biodistribution assays. Moreover, the in vivo therapeutic effects of PTX-loaded LPL (PTX-LPL) were observed, with an 80.84% inhibition rate of tumor growth and a 10.62% metastatic rate of tumor foci in lung tissue. Furthermore, the amounts of MMP-9 and neutrophils in the tumor as well as in the lung were greatly reduced with PTX-LPL. In summary, LPL may have potential applications in metastatic breast cancer therapy.

Development of an LC-MS/MS-based assay to determine artemitin in rat plasma and its application in a pharmacokinetic study.

Artemitin, a significant flavonol compound existing in Laggera pterodonta (DC.) Benth., Artemisia rupestris L, etc., is the subject of attention by researchers owing to its pharmacological activities (such as antioxidative, anti-inflammatory and antiviral). In this work, a highly sensitive and specific high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) assay combined with protein precipitation has been established and validated for determining artemitin concentration in rat plasma. Both artemitin and warfarin sodium (internal standard, IS) were separated on an Agela Venusil XBP Phenyl column through the isocratic elution mode of methanol-water containing 0.1% formic acid (80:20, v/v), at a flow rate of 0.4 mL/min. The MS/MS system was operated in a positive ion and ESI multiple reaction monitoring mode, and the multiple reaction monitoring transition was optimized as m/z 389.0 → 373.0 for artemitin and 309.2 → 163.0 for IS. The method showed good linearity in the range of 2.5-2000 ng/mL (R2 = 1.0000) and high sensitivity for artemitin with the lower limit of quantification of 2.5 ng/mL. The intra- and inter-day accuracies were 97.4-100.9 and 93.4-100.3%, respectively. The intra- and inter-day precisions were <4.8 and 6.5%, respectively. The extraction efficiency and absolute recovery were >66.5 and 71.3%, respectively. In addition, a good matrix effect of <9.5% was obtained. As a result, the method developed herein was successfully applied for the pharmacokinetic study of artemitin after an intravenous administration in rats.

Matrix metalloproteinase 2/9-triggered-release micelles for inhaled drug delivery to treat lung cancer: preparation and in vitro/in vivo studies.

BACKGROUND: Improvement in drug accumulation in the lungs through inhalation administration and high expression of MMP2 and MMP9 in lung tumors have both been widely reported. METHODS: MMP2/9-triggered-release micelles were constructed and in vitro and in vivo studies of inhalation administration against lung tumor carried out. Pluronic P123 (P123) was modified with GPLGIAGQ-NH2 (GQ8) peptide to obtain P123-GQ8 (PG). MMP2/9-triggered-release micelles were constructed using PG and succinylated gelatin (SG) and loading paclitaxel (Ptx). To study biodistribution of micelles, DiR encapsulated in micelles was dosed to rats via intravenous injection or inhalation before ex vivo imaging for detecting DiR quantity in lungs. And B16F10 lung cancer-bearing nude mice were chosen as animal models to evaluate in vivo efficacy of MMP2/9-triggered-release micelles. RESULTS: Ptx-release efficiency from PG-SG-Ptx micelles was MMP2/9-concentration-dependent. For A549 cells, PG-SG-Ptx cytotoxicity was significantly greater (P<0.001) compared to P123-Ptx. Aerosol inhalation was chosen as the method of administration. In biodistribution experiment, DiR quantity in lungs was 5.8%±0.4% of that in major organs, while the ratio was 38.8%±0.5% for inhalation. For B16F10 lung cancer-bearing nude mice, the efficacy of inhalation of PG-SG-Ptx was significantly higher (P<0.001) than Taxol inhalation and injected PG-SG-Ptx. Inhaled PG-SG-Ptx also significantly inhibited the expression of Pgp in lung cancer. CONCLUSION: Inhalation of MMP2/9-triggered-release micelles increased tumor sensitivity to chemotherapeutics and reduced the toxicity of chemotherapy to healthy lung cells, which has great potential in lung cancer therapy.

Blocking Autophagic Flux Enhances Iron Oxide Nanoparticle Photothermal Therapeutic Efficiency in Cancer Treatment.

Autophagy is a conservative eukaryotic pathway which plays a crucial role in maintaining cellular homeostasis, and dysfunction of autophagy is usually associated with pathological conditions. Recently, emerging reports have stressed that various types of nanomaterials and therapeutic approaches interfere with cellular autophagy process, which has brought up concerns to their future biomedical applications. Here, we present a study elaborating the relationships between autophagy and iron oxide nanoparticle (IONP)-mediated photothermal therapy in cancer treatment. Our results reveal that IONP photothermal effect could lead to autophagy induction in cancerous MCF-7 cells in a laser dose-dependent manner, and the inhibition of autophagy would enhance the photothermal cell killing by increasing cell apoptosis. In an MCF-7 xenograft model, cotreatment of autophagy inhibitor and IONP under laser exposure could promote the tumor inhibition rate from 43.26 to 68.56%, and the tumor immunohistochemistry assay of microtubule-associated protein 1-light chain 3 (LC3) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling also demonstrate augmentation in both autophagosomes accumulation and apoptosis in vivo. This work helps us to better understand the regulation of autophagy during IONP-mediated photothermal therapy and provides us with a potential combination therapeutic approach of autophagy modulators and photothermal agents.

Ambroxol enhances anti-cancer effect of microtubule-stabilizing drug to lung carcinoma through blocking autophagic flux in lysosome-dependent way.

Lung carcinoma has become a more and more serious health problem as platinum-based chemotherapy remains a limited benefit. Accumulating evidences indicate that autophagy plays a significant role in decreased curative effect and chemotherapy failure. Inhibition of autophagy can potentiate anti-proliferation effect and contribute to tumor regression in lung carcinoma. Here, we showed that the expectorant drug ambroxol (Ax) promoted autophagosomes accumulation by blocking late-stage autophagic flux in lung carcinoma cells. Furthermore, Ax treatment caused alkalization of lysosome and impaired lysosomal degradation capacity, which contributed to decreased autophagosomes-lysosomes fusion and interrupted normal cargo degradation. Ax potentiated cell-killing sensitivity of paclitaxel (PTX) and docetaxel (DTX), which had nothing to do with cell uptake but was associated with enhanced autophagy level. Moreover, Ax in combination with PTX exerted a significantly enhanced tumor-shrinking effect and prolonged survival time in subcutaneous and pulmonary metastatic tumor nude mice models. Considering the superiority of lung protection and excellent safety, Ax shows enormous translational potential and preponderance in clinical lung carcinoma therapy. Together, our findings suggested that the novel function of Ax, namely autophagy inhibition, resulted from alkalization and impaired degradation capacity of lysosome. The combination of Ax and PTX showed an enhanced cytotoxicity in vitro and improved satisfactory curative outcome in vivo. Our research provides a promising therapeutic strategy to lung carcinoma, which has clinical transformation potential and practical application value.