Optimal development of apoptotic cells-mimicking liposomes targeting macrophages.

Macrophages are multifunctional innate immune cells that play indispensable roles in homeostasis, tissue repair, and immune regulation. However, dysregulated activation of macrophages is implicated in the pathogenesis of various human disorders, making them a potential target for treatment. Through the expression of pattern recognition and scavenger receptors, macrophages exhibit selective uptake of pathogens and apoptotic cells. Consequently, the utilization of drug carriers that mimic pathogenic or apoptotic signals shows potential for targeted delivery to macrophages. In this study, a series of mannosylated or/and phosphatidylserine (PS) -presenting liposomes were developed to target macrophages via the design of experiment (DoE) strategy and the trial-and-error (TaE) approach. The optimal molar ratio for the liposome formulation was DOPC: DSPS: Chol: PEG-PE = 20:60:20:2 based on the results of cellular uptake and cytotoxicity evaluation on RAW 264.7 and THP-1 in vitro. Results from in vivo distribution showed that, in the DSS-induced colitis model and collagen II-induced rheumatoid arthritis model, PS-presenting liposomes (PS-Lipo) showed the highest accumulation in intestine and paws respectively, which holds promising potential for macrophage target therapy since macrophages are abundant at inflammatory sites and contribute to the progression of corresponding diseases. Organs such as the heart, liver, spleen, lung, and kidney did not exhibit histological alterations such as inflammation or necrosis when exposed to PC-presenting liposomes (PC-Lipo) or PS-Lipo. In addition, liposomes demonstrated hemobiocompatibility and no toxicity to liver or kidney for circulation and did not induce metabolic injury in the animals. Thus, the well-designed PS-Lipo demonstrated the most potential for macrophage target therapy.

Improved Synthesis of a Novel Biodegradable Tunable Micellar Polymer Based on Partially Hydrogenated Poly(ß-malic Acid-co-benzyl Malate).

Poly(benzyl malate) (PBM), together with its derivatives, have been studied as nanocarriers for biomedical applications due to their superior biocompatibility and biodegradability. The acquisition of PBM is primarily from chemical routes, which could offer polymer-controlled molecular weight and a unique controllable morphology. Nowadays, the frequently used synthesis from L-aspartic acid gives an overall yield of 4.5%. In this work, a novel synthesis route with malic acid as the initiator was successfully designed and optimized, increasing the reaction yield up to 31.2%. Furthermore, a crystalline form of PBM (PBM-2) that polymerized from high optical purity benzyl-ß-malolactonate (MLABn) was discovered during the optimization process. X-ray diffraction (XRD) patterns revealed that the crystalline PBM-2 had obvious diffraction peaks, demonstrating that its internal atoms were arranged in a more orderly manner and were different from the amorphous PBM-1 prepared from the racemic MLABn. The differential scanning calorimetry (DSC) curves and thermogravimetric curves elucidated the diverse thermal behaviors between PBM-1 and PBM-2. The degradation curves and scanning electron microscopy (SEM) images further demonstrated the biodegradability of PBM, which have different crystal structures. The hardness of PBM-2 implied the potential application in bone regeneration, while it resulted in the reduction of solubility when compared with PBM-1, which made it difficult to be dissolved and hydrogenated. The solution was therefore heated up to 75 °C to achieve benzyl deprotection, and a series of partially hydrogenated PBM was sequent prepared. Their optimal hydrogenation rates were screened to determine the optimal conditions for the formation of micelles suitable for drug-carrier applications. In summary, the synthesis route from malic acid facilitated the production of PBM for a shorter time and with a higher yield. The biodegradability, biosafety, mechanical properties, and adjustable hydrogenation widen the application of PBM with tunable properties as drug carriers.

Tissue-Engineered Trachea Consisting of Electrospun Patterned sc-PLA/GO- g-IL Fibrous Membranes with Antibacterial Property and 3D-Printed Skeletons with Elasticity.

In this study, a tissue-engineered trachea, consisting of multilevel structural electrospun polylactide (PLA) membranes enveloping 3D-printed thermoplastic polyurethane (TPU) skeletons, was developed to create a mechanically robust, antibacterial and bioresorbable graft for the tracheal reconstruction. The study design incorporated two distinct uses of stereocomplex PLA: patterned electrospun fibers to enhance tissue integration compared to the random layered fibers, meanwhile possessing good antibacterial property; and 3D-printed TPU scaffold with elasticity to provide external support and protection. Herein, ionic liquid (IL)-functioned graphene oxide (GO) was synthesized and presented enhanced mechanical and hydrophilicity properties. More interesting, antibacterial activity of the GO- g-IL modified PLA membranes were proved by Escherichia coli and Staphylococcus aureus, showing superior antibacterial effect compared to single GO or IL. The synergistic antibacterial effect could be related to that GO break cytomembrane of bacteria by its extremely sharp edges, while IL works by electrostatic interaction between its cationic structures and electronegative phosphate groups of bacteria membranes, leading to the loss of cell electrolyte and cell death. Hence, after L929 fibroblast cells were seeded on patterned fibrous membranes with phenotypic shape, further effective cell infiltration, cell proliferation and attachment were observed. In addition, the tissue-engineered trachea scaffolds were implanted into rabbit models. The in vivo result confirmed that the scaffolds with patterned membranes manifested favorable biocompatibility and promoted tissue regeneration.

MMP-2 sensitive poly(malic acid) micelles stabilized by π-π stacking enable high drug loading capacity.

Poly(ß-l-malic acid) (PMLA) together with its derivatives is an aliphatic polyester with superior bio-properties for anti-tumor drugs. In order to surmount the obstacles of low drug loading and rapid premature release during the circulation of polyester-based micelles, micelles based on poly(ß-benzyl malate)-b-polyethylene glycol (PBM-PEG) were developed in this study. The micelles had high drug loading capacity (>20 wt%) and held robust stability, owing to the π-π stacking interactions between polymer chains, and between the polymer and drug. Computer simulation also confirmed that there was the strongest binding free energy between PBMs, and PBM and doxorubicin (DOX), compared with other polyesters. A cell-penetrating moiety (TAT) was employed, and furthermore, a protective outer shell (PEG5k) was also introduced via a matrix metalloproteinase-2 (MMP-2) cleavable peptide. Before reaching the tumor site, the TAT peptide was shielded by long chain PEG, and the micelles showed low bioactivity. While at the tumor tissues where MMP-2 was highly expressed, the cleavage of the linker leads to the exposure of TAT, thus enhancing the cellular internalization. The desired therapeutic consequent was also observed, with no accompanying systemic toxicity detected. Our findings indicated that this MMP-2 sensitive PBM polymeric micelle would be a promising antitumor drug carrier with enhanced therapeutic effects.

Bacteria-activated chlorin e6 ionic liquid based on cation and anion dual-mode antibacterial action for enhanced photodynamic efficacy.

With the increase in antibiotic resistance, the development of new antibacterial agents is urgent. Photosensitizers with no detectable resistance are promising antibacterial agents. However, most photosensitizers are insoluble, structurally unstable and ineffective against Gram-negative bacteria due to their negatively charged cell wall that hinder their use. In this study, a novel bacteria-activated photosensitizer ionic liquid was designed and assembled to improve the solubility, stability and antibacterial ability of photodynamic therapy. The cation 1-vinyl-3-dodecyl imidazole has been designed, which has strong binding energy with the major constituent of the cell wall. The anion selected was chlorin e6 (Ce6) since it could respond to the acidic microenvironment of bacterial infection. The Ce6 ionic liquid (Ce6-IL) composed of 1-vinyl-3-dodecyl imidazole and Ce6 not only exhibited bacteria-activated ability because its cation could firmly bond with peptidoglycan in the cell wall, but also had excellent acid responsive ability due to the protonation reaction of COO- in its anion. The binding energy of the cation with peptidoglycan was calculated via molecular dynamics simulation, and the pH-responsive behavior of Ce6-IL was verified via HR-MS. The surface potential, mechanical property, morphology and uptake rate results indicated that the cation could destroy the cell wall and promote the anion Ce6 to enter the bacteria. Due to the dual-mode antibacterial action of its cation and anion, Ce6-IL was more effective against Gram-negative and Gram-positive bacteria than Ce6 alone and had wide-spectrum antibacterial ability. The in vitro studies showed that the IC50 of Ce6-IL against E. coli and S. aureus was reduced by 100 and 10 times, respectively. Furthermore, the in vivo studies indicated that Ce6-IL was more effective for eliminating bacterial infection and could accelerate wound healing. The compatibility test showed that Ce6-IL had low toxicity and exhibited excellent biocompatibility.

Enhanced Endocytic and pH-Sensitive Poly(malic acid) Micelles for Antitumor Drug Delivery.

Poly(ß-benzyl malate) (PBM), a derivative of poly(ß-malic acid) (PMLA), is a potential antitumor drug carrier due to its desirable biocompatibility and nontoxicity. In this study, micelles based on PBM-PEG polymers were prepared, which possessed several key features, including (i) micelle formation via self-assembly with a size of approximately 100 nm, (ii) π-π stacking interactions between the polymer chains and between the polymer and the drug, improving the stability of micelles and drug loading capacity (drug loading rate increased to 20 wt%), (iii) the cell penetrating peptide (TAT) was shielded by a long PEG chain before reaching the tumor site and exposed to tumor tissue, and (iv) high efficiency tumor uptake via exposure to TAT. At the site of a tumor, the extracellular pH level caused cleavage of the hydrazine bond, which led to the exposure of TAT on the polymeric micelles, thus enhancing cellular internalization. Then, the polymeric micelles disintegrated and DOX was released in response to the acidic pH in the lysosomal and endosomal compartments within the tumor cells. Both in vitro and in vivo efficacy studies indicated that this pH-sensitive PBM polymeric micelle is a promising antitumor drug carrier.

Preparation and biological evaluation of a novel pH-sensitive poly (ß-malic acid) conjugate for antitumor drug delivery.

Poly (ß­malic acid), referred to as PMLA, has been synthesized and introduced as a polymeric drug carrier due to its desirable biological properties. In the present study, a novel pH­sensitive polymer­drug conjugate based on PMLA, PMLA­Hz­doxorubicin (DOX), was prepared, and another conjugate, PMLA­ami­DOX, was synthesized as a comparison. The structures, conjugation efficiency, and drug release properties of the prodrugs were determined. The cytotoxicity and cell uptake were assessed using the HT1080 human fibrosarcoma cell line as an in vitro cell model. The release of DOX in the two conjugates were pH­dependent in PBS buffer at a pH of 5.6, 6.0, 6.8 and 7.4. The quantity of drug released increased with the decrease in pH, and PMLA­ami­DOX released twice as much as PMLA­Hz­DOX (12 h). The cytotoxicity of PMLA­Hz­DOX at pH 7.4 was lower than that of free DOX and increased with the decrease in pH, indicating that the cytotoxicity of PMLA­Hz­DOX was pH­sensitive. Flow cytometry and confocal experiments confirmed the efficiency of the PMLA­Hz­DOX conjugate. Therefore, bonding DOX to PMLA via an acid­sensitive hydrazone bond may be used to reduce its toxic side effects on normal tissues while responding to tumor pH and releasing the drug.

Optimal Design of Novel Functionalized Nanoconjugates Based on Polymalic Acid for Efficient Tumor Endocytosis with Enhanced Anticancer Activity.

To overcome the strong negative charge and improve the endocytosis of poly-ß-malic acid (PMLA) as a drug carrier, a pH-sensitive nanoconjugate of PMLA/hyd-PEG5000/PEG2000-TAT/DOX (PHPTD) was developed. The trans activator of transcription (TAT) modified with polyethylene glycol2000(PEG2000) was conjugated with the PMLA backbone which improved the endocytosis of PMLA. PEG5000 was utilized to shield TAT by a pH-sensitive hydrazone (Hyd) bond. In order to decrease the potential risk of accelerated blood clearance (ABC) phenomenon by anti-PEG IgM, the minimal content of TAT for penetrating tumor cells and the optimal protecting layer density of PEG5000 were screened. The result showed that 0.3 mol% TAT was enough to efficiently improve cellular uptake of PMLA (30 kda). The cytotoxicity and the 1H-NMR results indicated that 3.6 mol% PEG5000-modified nanoconjugates could shield 0.3 mol% TAT. The antitumor effect in breast cancer cells (MDA-MB-231) in tumor-bearing BALB/C mice demonstrated that this nanoconjugates exhibits high therapeutic efficiency in artificial solid tumors and low toxicity to normal tissues. It is indicated that TAT could be hidden in the long chain of PEG5000 at a neutral pH, when arrival to the tumor extracellular microenvironment, PEG5000 was cleaved from the nanoconjugates through the hydrazone bond due to the acidic tumor environment. Then, TAT was exposed, allowing the nanoconjugates to be transported into tumor cells. Our findings provide important and detailed information regarding the optimal content of TAT and the shielded density of PEG5000 and reveal their abilities of tumor penetration and potential for the efficient drug carrier.

Dual-pH Sensitive Charge-reversal Nanocomplex for Tumor-targeted Drug Delivery with Enhanced Anticancer Activity.

Poly(ß-L-malic acid) (PMLA), a natural aliphatic polyester, has been proven to be a promising carrier for anti-cancer drugs. In spite of excellent bio-compatibility, the application of PMLA as the drug carrier for cancer therapy is limited by its low cellular uptake efficiency. The strong negative charge of PMLA impedes its uptake by cancer cells because of the electrostatic repulsion. In this study, a dual pH-sensitive charge-reversal PMLA-based nanocomplex (PMLA-PEI-DOX-TAT@PEG-DMMA) was developed for effective tumor-targeted drug delivery, enhanced cellular uptake, and intracellular drug release. The prepared nanocomplex showed a negative surface charge at the physiological pH, which could protect the nanocomplex from the attack of plasma proteins and recognition by the reticuloendothelial system, so as to prolong its circulation time. While at the tumor extracellular pH 6.8, the DMMA was hydrolyzed, leading to the charge reversal and exposure of the TAT on the polymeric micelles, thus enhancing the cellular internalization. Then, the polymeric micelles underwent dissociation and drug release in response to the acidic pH in the lyso/endosomal compartments of the tumor cell. Both in vitro and in vivo efficacy studies indicated that the nanocomplex significantly inhibited the tumor growth while the treatment showed negligible systemic toxicity, suggesting that the developed dual pH-sensitive PMLA-based nanocomplex would be a promising drug delivery system for tumor-targeted drug delivery with enhanced anticancer activity.

Preparation of Two Types of Polymeric Micelles Based on Poly(ß-L-Malic Acid) for Antitumor Drug Delivery.

Polymeric micelles represent an effective delivery system for poorly water-soluble anticancer drugs. In this work, two types of CPT-conjugated polymers were synthesized based on poly(ß-L-malic acid) (PMLA) derivatives. Folic acid (FA) was introduced into the polymers as tumor targeting group. The micellization behaviors of these polymers and antitumor activity of different self-assembled micelles were investigated. Results indicate that poly(ethylene glycol)-poly(ß-L-malic acid)-campotothecin-I (PEG-PMLA-CPT-I, P1) is a grafted copolymer, and could form star micelles in aqueous solution with a diameter of about 97 nm, also that PEG-PMLA-CPT-II (P2) is an amphiphilic block copolymer, and could form crew cut micelles with a diameter of about 76 nm. Both P1 and P2 micelles could improve the cellular uptake of CPT, especially the FA-modified micelles, while P2 micelles showed higher stability, higher drug loading efficiency, smaller size, and slower drug release rate than that of P1 micelles. These results suggested that the P2 (crew cut) micelles possess better stability than that of the P1 (star) micelles and might be a potential drug delivery system for cancer therapy.

Dual loading miR-218 mimics and Temozolomide using AuCOOH@FA-CS drug delivery system: promising targeted anti-tumor drug delivery system with sequential release functions.

BACKGROUND: Dual loading drug delivery system with tumor targeting efficacy and sequential release function provides a promising platform for anticancer drug delivery. Herein, we established a novel AuCOOH@FACS nanogel system for co-delivery miR-218 mimics (as bio-drug) and Temozolomide(as chemo-drug). METHODS: DLS and TEM were employed to determine the characteristics of particles and nanogels. The cell viability was calculated for study synergistic effect of both drugs coadministration and in nanogel forms. The amounts of Au uptake were measured by ICP-MS in cell and tumors to quantify the targeting drug delivery efficacy. Tumor weight and mice weight were investigated to study the targeting antitumor efficacy of nanogel system. RESULTS: The results revealed that using AuCOOH@FACS nanogel as delivery vehicles, drugs could be targeting delivery to tumor site, the intracellular uptake is enhanced to a greater extent, and significant antitumor efficacy is fold increase compared with free drug administration group, without noticeable system cytotoxicity. CONCLUSIONS: This system offers an efficient approach to cancer therapy and holds significant potential to improve the treatment of cancer in the future.

Preparation of poly(ß-L-malic acid)-based charge-conversional nanoconjugates for tumor-specific uptake and cellular delivery.

In this study, a multifunctional poly(ß-L-malic acid)-based nanoconjugate with a pH-dependent charge conversional characteristic was developed for tumor-specific drug delivery. The short branched polyethylenimine-modified poly(ß-L-malic acid) (PEPM) was first synthesized. Then, the fragment HAb18 F(ab')2 and 2,3-dimethylmaleic anhydride were covalently attached to the PEPM to form the nanoconjugate, HDPEPM. In this nanoconjugate, the 2,3-dimethylmaleic anhydride, the shielding group, could shield the positive charge of the conjugate at pH 7.4, while it was selectively hydrolyzed in the tumor extracellular space (pH 6.8) to expose the previously-shielded positive charge. To study the anticancer activity, the anticancer drug, doxorubicin, was covalently attached to the nanoconjugate. The doxorubicin-loaded HDPEPM nanoconjugate was able to efficiently undergo a quick charge conversion from -11.62 mV to 9.04 mV in response to the tumor extracellular pH. The electrostatic interaction between the positively charged HDPEPM nanoconjugates and the negatively charged cell membrane significantly enhanced their cellular uptake, resulting in the enhanced anticancer activity. Also, the tumor targetability of the nanoconjugates could be further improved via the fragment HAb18 F(ab')2 ligand-receptor-mediated tumor cell-specific endocytosis.

Nanobubble-Affibody: Novel ultrasound contrast agents for targeted molecular ultrasound imaging of tumor.

Nanobubbles (NBs), as novel ultrasound contrast agents (UCAs), have attracted increasing attention in the field of molecular ultrasound imaging for tumors. However, the preparation of uniform-sized NBs is considered to be controversial, and poor tumor selectivity in in vivo imaging has been reported. In this study, we fabricated uniform nano-sized NBs (478.2 ± 29.7 nm with polydispersity index of 0.164 ± 0.044, n = 3) using a thin-film hydration method by controlling the thickness of phospholipid films; we then conjugated the NBs with Affibody molecules to produce nano-sized UCAs referred to as NB-Affibody with specific affinity to human epidermal growth factor receptor type 2 (HER2)-overexpressing tumors. NB-Affibody presented good ultrasound enhancement, demonstrating a peak intensity of 104.5 ± 2.1 dB under ultrasound contrast scanning. Ex vivo experiments further confirmed that the NB-Affibody conjugates were capable of targeting HER2-expressing tumor cells in vivo with high affinity. The newly prepared nano-sized NB-Affibody conjugates were observed to be novel targeted UCAs for efficient and safe specific molecular imaging and may have potential applications in early cancer quantitative diagnosis and targeted therapy in the future.

GNPs-CS/KGM as hemostatic first aid wound dressing with antibiotic effect: in vitro and in vivo study.

Ideal wound dressing materials should create a good healing environment, with immediate hemostatic effects and antimicrobial activity. In this study, chitosan/konjac glucomannan (CS/KGM) films embedded with gentamicin-loaded poly(dex-GMA/AAc) nanoparticles (giving GNP-CS/KGM films) were prepared as novel wound dressings. The results revealed that the modified CS/KGM films could be used as effective wound dressings and had significant hemostatic effects. With their microporous structure, the films could effectively absorb water from blood and trap blood cells. The gentamicinloaded poly(dex-GMA/AAc) nanoparticles (GNPs) also further promoted blood clotting, with their favorable water uptake capacity. Thus, the GNP-CS/KGM films had wound healing and synergistic effects that helped to stop bleeding from injuries, and also showed good antibiotic abilities by addition of gentamicin to the NPs. These GNPCS/KGM films can be considered as promising novel biodegradable and biocompatible wound dressings with hemostatic capabilities and antibiotic effects for treatment of external bleeding injuries.