Effectiveness of early glucocorticoids in myasthenia gravis: a retrospective cohort study.

Purpose: This study aimed to clarify the effect of early glucocorticoid (GC) application on achieving minimal manifestation (MM) status or better in the treatment of myasthenia gravis (MG) in the early clinical phase. Methods: A retrospective analysis was performed using data from 336 patients with MG who received GC therapy from January 2015 to September 2022 in the Zhengzhou University Henan Institute of Medical and Pharmaceutical Sciences Myasthenia Gravis Biobank (ZMB). Patients were divided into two groups: the early mono-GC group (treated with GC within 6 months of MG onset) and the delayed mono-GC group. Results: Kaplan-Meier analysis showed that the early mono-GC group achieved MM status earlier and more frequently than the delayed mono-GC group (log-rank test, p = 0.0082; hazard ratio [HR], 1.66; p = 0.011). The early mono-GC group had a lower maintenance oral GC dose than the delayed mono-GC group. In multivariate Cox regression analysis, early mono-GC (HR, 1.50; p = 0.043), early-onset MG (EOMG) (HR, 1.74; p = 0.034), and ocular MG (OMG) (HR, 1.90; p = 0.007) were associated with MM status or better. In conclusion, early mono-GC, EOMG, and OMG were positive predictors of treatment goals. In EOMG, OMG, and acetylcholine receptor antibody-positive MG (AChR-MG) subgroups, the maintenance oral GC doses in the early mono-GC group were significantly lower than the doses in the delayed mono-GC group (p < 0.05). Conclusion: Early intervention with GC led to better long-term outcomes and reduced the necessary maintenance dose of oral GC for patients with MG. EOMG and OMG were positive predictors of MM status or better with mono-GC.

Utilizing G2/M retention effect to enhance tumor accumulation of active targeting nanoparticles.

In recent years, active targeting strategies by ligand modification have emerged to enhance tumor accumulation of NP, but their clinical application was strictly restricted due to the complex preparation procedures, poor stability and serious toxicity. An effective and clinical translational strategy is required to satisfy the current problems. Interestingly, the internalization of NP is intimately related with cell cycle and the expression of receptors is not only related with cancer types but also cell cycle progression. So the cellular uptake of ligand modified NP may be related with cell cycle. However, few investigations were reported about the relationship between cell cycle and the internalization of ligand modified NP. Herein, cellular uptake of folic acid (FA) modified NP after utilizing chemotherapeutic to retain the tumor cells in G2/M phase was studied and a novel strategy was designed to enhance the active targeting effect. In our study, docetaxel (DTX) notably synchronized cells in G2/M phase and pretreatment with DTX highly improved in vitro and in vivo tumor cell targeting effect of FA decorated NP (FANP). Since FA was a most common used tumor active targeting ligand, we believe that this strategy possesses broader prospects in clinical application for its simplicity and effectiveness.

Significantly enhanced tumor cellular and lysosomal hydroxychloroquine delivery by smart liposomes for optimal autophagy inhibition and improved antitumor efficiency with liposomal doxorubicin.

Hydroxychloroquine (HCQ) inhibits autophagy and therefore can sensitize some cancer cells to chemotherapy, but the high doses required limit its clinical use. Here we show that loading HCQ into liposomes (HCQ/Lip) decorated with a pH-sensitive TH-RGD targeting peptide (HCQ/Lip-TR) can concentrate HCQ in B16F10 tumor cells and lysosomes. HCQ/Lip-TR was efficiently internalized as a result of its ability to bind ITGAV-ITGB3/integrin αvß3 receptors highly expressed on the tumor cell surface and to undergo charge reversal from anionic at pH 7.4 to cationic at pH 6.5. Studies in vitro at pH 6.5 showed that the intracellular HCQ concentration was 35.68-fold higher, and lysosomal HCQ concentration 32.22-fold higher, after treating cultures with HCQ/Lip-TR than after treating them with free HCQ. The corresponding enhancements observed in mice bearing B16F10 tumors were 15.16-fold within tumor cells and 14.10-fold within lysosomes. HCQ/Lip-TR was associated with milder anemia and milder myosuppressive reductions in white blood cell and platelet counts than free HCQ, as well as less accumulation in the small intestine, which may reduce risk of intestinal side effects. In addition, co-delivering HCQ/Lip-TR with either free doxorubicin (DOX) or liposomal DOX improved the ability of DOX to inhibit tumor growth. Biochemical, electron microscopy and immunofluorescence experiments confirmed that HCQ/Lip-TR blocked autophagic flux in tumor cells. Our results suggest that loading HCQ into Lip-TR liposomes may increase the effective concentration of the inhibitor in tumor cells, allowing less toxic doses to be used.

Integrin-mediated active tumor targeting and tumor microenvironment response dendrimer-gelatin nanoparticles for drug delivery and tumor treatment.

Due to the high morbidity and mortality of cancer, it has become an urgent matter to develop an effective and a safe treatment strategy. Nanoparticles (NP) based drug delivery systems have gained much attention nowadays but they faced a paradoxical issue in delivering drugs into tumors: NP with large size were characterized with weak tumor penetration, meanwhile NP with small size resulted in poor tumor retention. To solve this problem, we proposed a multistage drug delivery system which could intelligently shrink its size from large size to small size in the presence of matrix metalloproteinase-2 (MMP-2) which were highly expressed in tumor tissues, therefore the multistage system could benefit from its large size for better retention effect in tumor and then shrunk to small size to contribute to better penetration efficiency. The multistage drug delivery system, RGD-DOX-DGL-GNP, was constructed by 155.4nm gelatin NP core (the substrate of MMP-2) and surface decorated with doxorubicin (DOX) and RGD peptide conjugated dendritic poly-l-lysine (DGL, 34.3nm in diameter). In vitro, the size of multistage NP could effectively shrink in the presence of MMP-2. Thus, the RGD-DOX-DGL-GNP could penetrate deep into tumor spheroids. In vivo, this multistage drug delivery system showed higher tumor retention and deeper penetration than both DOX-DGL and DOX-GNP. Consequently, RGD-DOX-DGL-GNP successfully combined the advantages of dendrimers and GNP in vivo, resulting in an outstanding anti-tumor effect. In conclusion, the multistage drug delivery system could intelligently shrink from large size to small size in the tumor microenvironment and displayed better retention and penetration efficiency, making it an impressing system for cancer treatment.

Peptide mediated active targeting and intelligent particle size reduction-mediated enhanced penetrating of fabricated nanoparticles for triple-negative breast cancer treatment.

Triple-negative breast cancer (TNBC) is one of the most invasively malignant human cancers and its incidence increases year by year. Effective therapeutics against them needs to be developed urgently. In this study, a kind of angiopep-2 modified and intelligently particle size-reducible NPs, Angio-DOX-DGL-GNP, was designed for accomplishing both high accumulation and deep penetration within tumor tissues. On one hand, for improving the cancerous targeting efficiency of NPs, angiopep-2 was anchored on the surface of NPs to facilitate their accumulation via binding with low density lipoprotein-receptor related protein (LRP) overexpressed on TNBC. On the other hand, for achieving high tumor retention and increasing tumor penetration, an intelligently particle size-reducible NPs were constructed through fabricating gelatin NPs (GNP) with doxorubicin (DOX) loaded dendrigraft poly-lysine (DGL). In vitro cellular uptake and ex-vivo imaging proved the tumor targeting effect of Angio-DOX-DGL-GNP. Additionally, the degradation of large-sized Angio-DOX-DGL-GNP by matrix metalloproteinase-2 (MMP-2) led to the size reduction from 185.7 nm to 55.6 nm. More importantly, the penetration ability of Angio-DOX-DGL-GNP after incubation with MMP-2 was dominantly enhanced in tumor spheroids. Due to a combinational effect of active targeting and deep tumor penetration, the tumor growth inhibition rate of Angio-DOX-DGL-GNP was 74.1% in a 4T1 breast cancer bearing mouse model, which was significantly higher than other groups. Taken together, we successfully demonstrated a promising and effective nanoplatform for TNBC treatment.

Matrix metalloproteinase-sensitive size-shrinkable nanoparticles for deep tumor penetration and pH triggered doxorubicin release.

Nanocarriers are widely used for delivering drugs to tumors and are progressing in a stable trend. The enhanced permeability and retention (EPR) effect has been a key rationale for the development of stimulus-responsive nanocarriers to solid tumor. In this study, we developed a kind of novel nanocarrier, G-AuNPs-DOX-PEG, which was constructed with shrinkable gelatin nanoparticles coated, doxorubicin (DOX) tethered gold nanoparticles and long chain polyethylene glycol (PEG). The particle size of G-AuNPs-DOX-PEG was 186.5 nm with a zeta potential of -4.21 mV and the DOX loading capacity was 9.22%. In vitro, the G-AuNPs-DOX-PEG could be degraded by MMP-2 proteins with a size shrink from 186.5 nm to 59.3 nm. The release of DOX from G-AuNPs-DOX-PEG was in a pH- and time-dependent manner. At pH 5.0, the release of DOX was much quicker than that at high pH value and the cumulative release rate of DOX from G-AuNPs-DOX-PEG was approach 90.9%. Cellular uptake demonstrated that G-AuNPs-DOX-PEG could be internalized via the endosome-mediated pathway. Tumor spheroid penetration and collagen gel diffusion showed G-AuNPs-DOX-PEG with pre-incubation with MMP-2 could significantly enhance its penetrating efficiency. In vivo and ex vivo imaging exhibit that G-AuNPs-DOX-PEG could distribute into 4T1 and B16F10 tumor at a highest intensity. Correspondingly, 4T1 and B16F10 tumor bearing mice treated with G-AuNPs-DOX-PEG displayed the lowest tumor growth rate. In summary, the tumor microenvironment sensitive size-shrinkable G-AuNPs-DOX-PEG could deliver into deep tumor region and then release DOX, resulting in a best anti-tumor effect.

Tumor-targeted paclitaxel delivery and enhanced penetration using TAT-decorated liposomes comprising redox-responsive poly(ethylene glycol).

To combine the advantage of poly(ethylene gylcol) (PEG) for longer circulation and cell-penetrating peptides (CPPs) for efficient cellular uptake, paclitaxel (PTX)-loaded liposomes functionalized with TAT, the most frequently used CPP, and cleavable PEG via a redox-responsive disulfide linker (PTX-C-TAT-LP) were successfully developed here. Under physiological conditions, TAT was shielded by PEG layer and liposomes exhibited a long blood circulation. At tumor site, PEG could be detached in the presence of exogenous reducing agent [glutathione (GSH)] and TAT was exposed to facilitate cell internalization. In the presence of GSH, the liposomal vesicle C-TAT-LP showed increased cellular uptake and improved three-dimensional tumor spheroids penetration in vitro compared with analogous stable shielded liposomes. C-TAT-LP achieved enhanced tumor distribution and demonstrated superior delivery efficiency in vivo. PTX-C-TAT-LP with GSH strongly inhibited the proliferation of murine melanoma B16F1 tumor cells in vitro and in vivo with the tumor inhibition rate being 69.4% on B16F1-bearing mice. In addition, the serum aspartate transaminase level, alanine transaminase level, and creatine kinase level were almost completely within normal range in the PTX-C-TAT-LP with GSH group, revealing PTX-C-TAT-LP with GSH had no obvious drug-related adverse events for liver and heart. Taken together, C-TAT-LP is a promising tumor-targeting drug carrier.

Tumor microenvironment sensitive doxorubicin delivery and release to glioma using angiopep-2 decorated gold nanoparticles.

Glioma is still hard to be treated due to their complex microenvironment. In this study, a gold nanoparticle-based delivery system was developed. The system, An-PEG-DOX-AuNPs, was loaded with doxorubicin (DOX) through hydrazone, an acid-responsive linker, and was functionalized with angiopep-2, a specific ligand of low density lipoprotein receptor-related protein-1 (LRP1), which could mediate the system to penetrate blood brain barrier and target to glioma cells. The particle size of An-PEG-DOX-AuNPs was 39.9 nm with a zeta potential of -19.3 mV, while the DOX loading capacity was 9.7%. In vitro, the release of DOX from DOX-AuNPs was pH-dependent. At lower pH values, especially 5.0 and 6.0, release of DOX was much quicker than that at pH 6.8 and 7.4. After coating with PEG, the acid-responsive release of DOX from PEG-DOX-AuNPs was almost the same as that from DOX-AuNPs. Cellular uptake study showed obviously higher intensity of intracellular An-PEG-DOX-AuNPs compared with PEG-DOX-AuNPs. In vivo, An-PEG-DOX-AuNPs could distribute into glioma at a higher intensity than that of PEG-DOX-AuNPs and free DOX. Correspondingly, glioma-bearing mice treated with An-PEG-DOX-AuNPs displayed the longest median survival time, which was 2.89-fold longer than that of saline. In conclusion, An-PEG-DOX-AuNPs could specifically deliver and release DOX in glioma and significantly expand the median survival time of glioma-bearing mice.

[Preparation of cell penetrating peptide TAT and cleavable PEGco-modified liposomes loaded with paclitaxel and its in vitro apoptosis assay].

The preparation method, serum stability, efficiency of cellular uptake and apoptosis induction of the cell penetrating peptide TAT and cleavable PEG co-modified liposomes loaded with paclitaxel (C-TAT-Lipo) were investigated. The best preparation procedure was performed by orthogonal test based on single factor screening method. First, the paclitaxel (PTX)-loaded liposomes were prepared by filming-rehydration method, evaluated with entrapment efficiency and polydispersity index. The morphology of C-TAT-Lipo was characterized by transmission electron microscopy. Turbidity variations were monitored in the presence of fetal bovine serum (FBS) to evaluate the serum stability of the liposomes developed here. Next, the efficiency of cellular uptake of different Rho-PE-labeled liposomes on B16F1 cells in vitro was evaluated by confocal laser scanning microscopy (CLSM) and flow cytometry. The quantitative analysis of apoptosis induced by different PTX-loaded liposomes was performed by Annexin V-FITC/PI double staining. The optimal formulation was as follows: Chol : lipid: 1 : 8 (molar ratio); drug : lipid: 1 : 40 (mass ratio); lipid concentration: 3 mmol x L(-1); temperature of hydration: 25 degrees C. The mean size and polydispersity index of C-TAT-Lipo were about (97.97 +/- 3.68) nm and 0.196 +/- 0.037, the zeta potential was (-0.89 +/- 0.45) mV, the entrapment efficiency of paclitaxel was (90.16 +/- 1.53)%. The particle sizes did not exhibit significant variations in 50% FBS over 24 h at 37 degrees C. The efficiency of cellular uptake of the C-TAT-Lipo increased 1.40 fold following the cleavage of PEG. Apoptosis analysis showed 59.3% increase of the apoptosis and necrosis profile of C-TAT-Lipo after the detachment of PEG shells, which was markedly higher than that of N-TAT-LP with or without glutathione and SL, respectively. The results indicate that the C-TAT-Lipo is successfully prepared by filming-rehydration method and shows significant antitumor activities.

Pretreatment with chemotherapeutics for enhanced nanoparticles accumulation in tumor: the potential role of G2 cycle retention effect.

Ligands were anchored onto nanoparticles (NPs) to improve the cell internalization and tumor localization of chemotherapeutics. However, the clinical application was shadowed by the complex preparation procedure and the immunogenicity and poor selectivity and stability of ligands. In this study, a novel strategy was developed to elevate the tumor cellular uptake and tumor localization of NPs utilizing the G2/M phase retention effect of docetaxel, one of the most common chemotherapeutics. Results showed pretreatment with docetaxel could effectively arrest cells in G2/M phase, leading to an enhanced cell uptake of NPs, which may be caused by the facilitated endocytosis of NPs. In vivo imaging and slice distribution also demonstrated the pretreatment with docetaxel improved the localization of NPs in tumor. This strategy can be easily transferred to clinical for cancer management. Combination chemotherapeutics injections with commercial nano-drugs may result in better antitumor effect than the administration of a single drug.