Self-assembled polymeric nanocarrier-mediated co-delivery of metformin and doxorubicin for melanoma therapy

Malignant melanoma is a life-threatening form of skin cancer with a low response rate to single-agent chemotherapy. Although combined therapies of metformin (MET) and doxorubicin (DOX) are effective in treating a variety of cancers, including breast cancer, their different physicochemical properties and administration routines reduce the effective co-accumulation of both drugs in tumors. Nanoparticles (NPs) have been demonstrated to potentially improve drug delivery efficiency in cancer therapy of, for example, liver and lung cancers. Hence, in this study, we prepared pH-sensitive, biocompatible, tumor-targeting NPs based on the conjugation of biomaterials, including sodium alginate, cholesterol, and folic acid (FCA). As expected, since cholesterol and folic acid are two essentials, but insufficient, substrates for melanoma growth, we observed that the FCA NPs specifically and highly effectively accumulated in xenograft melanoma tumors. Taking advantage of the FCA NP system, we successfully co-delivered a combination of MET and DOX into melanoma tumors to trigger pyroptosis, apoptosis, and necroptosis (PANoptosis) of the melanoma cells, thus blocking melanoma progression. Combined, the establishment of such an FCA NP system provides a promising vector for effective drug delivery into melanoma and increases the possibility and efficiency of drug combinations for cancer treatment.     

Amphiphilic dendritic nanomicelle-mediated co-delivery of 5-fluorouracil and doxorubicin for enhanced therapeutic efficacy

Combination cancer therapy has attracted considerable attention due to its enhanced antitumor efficacy and reduced toxicity granted by synergistic effects over monotherapy. The application of nanotechnology is expected to achieve coencapsulation of multiple anticancer agents with enhanced therapeutic efficacy. Herein, a unique nanomicelle based on amphiphilic dendrimer (AmD) consisting of a hydrophilic polyamidoamine dendritic shell and a hydrophobic polylactide core is developed for effectively loading and shuttling 5-fluorouracil (5-Fu) and doxorubicin (Dox). The yielded drug-encapsulated dendritic nanomicelle (5-Fu/Dox-DNM) has a modest average size of 68.6?±?3.3?nm and shows pH-sensitive drug release manner. The parallel activity of 5-Fu and Dox show synergistic anticancer efficacy. The IC₅₀ value of 5-Fu/Dox-DNM toward human breast cancer (MDA-MB-231) cells was 0.25?μg/mL, presenting an 11.2-fold and 6.1-fold increase in cytotoxicity compared to Dox-DNM and 5-Fu-DNM, respectively. Furthermore, 5-Fu/Dox-DNM significantly inhibits the progression of tumor growth in the MDA-MB-231 xenograft tumor mice model. In conclusion, we have demonstrated that our AmD-based combination therapeutic system has promising potential to open an avenue for coencapsulation of multiple chemotherapeutic agents to promote superior anticancer effect.     

Proteomic analysis of serum-derived extracellular vesicles in ankylosing spondylitis patients

BackgroundAnkylosing spondylitis (AS) is a chronic inflammatory disease, whose pathogenesis is still unclear. Many studies show the proteins in extracellular vesicle (EVs) would change regularly in many diseases. The study aims to explore the proteins contents of serum-derived EVs in AS patients.MethodsEVs were separated by ExoQuick^TM kit. The protein profiles of AS patients and healthy subjects were analyzed by Label-free-liquid chromatography mass spectrometry (LC-MS/MS) technology. Enzyme-linked immunosorbent assay (ELISA) was used to verify the levels of the differently expressed proteins. Receiver operation characteristic (ROC) curves and bioinformatic analysis were conducted.ResultsSix hundred and ten serum-derived EVs proteins from AS patients were detected. Seventy-three diferentially expressed proteins were found in AS group, compared with healthy subjects. Of these, 31 proteins were up-regulated in AS group, while 42 proteins were down-regulated. ELISA result showed that EVs-derived serum amyloid A-1 (SAA1) was higher in AS group, which was consistent with the Label-free-LC-MS/MS data. ROC curves result revealed that the area under curve (AUC) value of EVs-derived SAA1 for AS was 0.768 (0.652–0.885). Bioinformatic analysis revealed that the differently expressed proteins in AS group were significantly involved in “complement and coagulation cascades”, “staphylococcus aureus infection”, “systemic lupus erythematosus” and “PI3K-Akt signaling pathway”.ConclusionsThe protein profiles of serum-derived EVs in AS patients and healthy subjects were different. EVs-derived SAA1 may be a potential biomarkes of AS. The function analysis indicated that the differentially expressed proteins may potentially participate in immune response.     

共载阿霉素和依克立达的PLGA纳米粒的制备及表征

目的 建立药物测定方法,并制备共载阿霉素和依克立达的PLGA纳米粒。方法 利用紫外分光光度法（UV）和高效液相色谱法（HPLC）分别建立阿霉素和依克立达的测定方法;采用纳米沉淀法制备共载纳米粒,通过调节两药的投药比,优化处方,考察纳米粒的粒径、形态、包封率、载药量以及体外释放。结果 阿霉素在1~40 μg/ml浓度范围内线性关系良好,标准曲线回归方程为A=0.021C+0.002,r=0.999 5; 依克立达在0.5~100 μg/ml浓度范围内线性关系良好,标准曲线回归方程为A=120 742.462 6C+1 974.570 4,r=1.000 0;通过处方优化,共载纳米粒的粒径约为50 nm,分布均一,呈圆形,阿霉素和依克立达的包封率分别为56.58%、51.66%,载药量分别为1.48%、1.85%,两药摩尔比约为1：1;体外释放缓慢。结论 分别建立了方便快捷、结果准确、重复性好的阿霉素和依克立达的检测方法,并且制备了分散性好、粒径较小的纳米粒,为后续实验提供基础。     

Trends in the biological functions and medical applications of extracellular vesicles and analogues

Natural extracellular vesicles (EVs) play important roles in many life processes such as in the intermolecular transfer of substances and genetic information exchanges. Investigating the origins and working mechanisms of natural EVs may provide an understanding of life activities, especially regarding the occurrence and development of diseases. Additionally, due to their vesicular structure, EVs (in small molecules, nucleic acids, proteins, etc.) could act as efficient drug-delivery carriers. Herein, we describe the sources and biological functions of various EVs, summarize the roles of EVs in disease diagnosis and treatment, and review the application of EVs as drug-delivery carriers. We also assess the challenges and perspectives of EVs in biomedical applications.     

Preserving extracellular vesicles for biomedical applications: consideration of storage stability before and after isolation

Extracellular vesicles (EVs) are nanovesicles released by various cell types. EVs are known for cell-to-cell communications and have potent biological activities. Despite great progress in recent years for studies exploring the potentials of EVs for early disease detection, therapeutic application and drug delivery, determination of the favorable storage conditions of EVs has been challenging. The understanding of the impact of storage conditions on EVs before and after isolation is still limited. Storage may change the size, number, contents, functions, and behaviors of EVs. Here, we summarized current studies about the stability of EVs in different conditions, focusing on temperatures, durations, and freezing and thawing cycles. –80 °C seems to remain the most favorable condition for storage of biofluids and isolated EVs, while isolated EVs may be stored at 4 °C shortly. Lyophilization is promising for storage of EV products. Challenges remain in the understanding of storage-mediated change in EVs and in the development of advanced preservation techniques of EVs.     

Circulating extracellular vesicles as a potential source of new biomarkers of drug-induced liver injury

Like most cell types, hepatocytes constantly produce extracellular vesicles (EVs) such as exosomes and microvesicles that are released into the circulation to transport signaling molecules and cellular waste. Circulating EVs are being vigorously explored as biomarkers of diseases and toxicities, including drug-induced liver injury (DILI). Emerging data suggest that (a) blood-borne EVs contain liver-specific mRNAs and microRNAs (miRNAs), (b) the levels can be remarkably elevated in response to DILI, and (c) the increases correlate well with classical measures of liver damage. The expression profile of mRNAs in EVs and the compartmentalization of miRNAs within EVs or other blood fractions were found to be indicative of the offending drug involved in DILI, thus providing more informative assessment of liver injury than using alanine aminotransferase alone. EVs in the urine and cell culture medium were also found to contain proteins or mRNAs that were indicative of DILI. However, major improvements in EV isolation methods are needed for the discovery, evaluation, and quantification of possible DILI biomarkers in circulating EVs.     

Exosomes: Breast cancer-derived extracellular vesicles; recent key findings and technologies in disease progression,diagnostics, and cancer targeting

Breast cancer is one of the most frequently diagnosed types of cancer in women. Metastasis, particularly to the lungs and brain, increases mortality in breast cancer patients. Recently, breast cancer-related exosomes have received significant attention because of their key role in breast cancer progression. As a result, numerous exosome-based therapeutic tools for diagnosis and treatment have been developed, and their biological and chemical mechanisms have been explored. This review summarizes up-to-date advanced key findings and technologies in breast cancer progression, diagnostics, and targeting. We focused on recent research on the basic biology of exosomes and disease-related exosomal genes and proteins, as well as their signal transduction in cell-to-cell communications, diagnostic markers, and exosome-based antibreast cancer technologies. We also paid special attention to technologies employing exosomes modified with functional peptides for the targeted delivery of therapeutic and diagnostic agents.     

Cell-derived biomimetic nanocarriers for targeted cancer therapy: cell membranes and extracellular vesicles

Nanotechnology provides synthetic carriers for cancer drug delivery that protect cargos from degradation, control drug release and increase local accumulation at tumors. However, these non-natural vehicles display poor tumor targeting and potential toxicity and are eliminated by the immune system. Recently, biomimetic nanocarriers have been widely developed based on the concept of ‘mimicking nature.’ Among them, cell-derived biomimetic vehicles have become the focus of bionics research because of their multiple natural functions, such as low immunogenicity, long circulation time and targeting ability. Cell membrane-coated carriers and extracellular vesicles are two widely used cell-based biomimetic materials. Here, this review summarizes the latest progress in the application of these two biomimetic carriers in targeted cancer therapy. Their properties and performance are compared, and their future challenges and development prospects are discussed.     

Innovative strategy for treatment of lung cancer: targeted nanotechnology-based inhalation co-delivery of anticancer drugs and siRNA

A tumor targeted mesoporous silica nanoparticles (MSN)-based drug delivery system (DDS) was developed for inhalation treatment of lung cancer. The system was capable of effectively delivering inside cancer cells anticancer drugs (doxorubicin and cisplatin) combined with two types of siRNA targeted to MRP1 and BCL2 mRNA for suppression of pump and nonpump cellular resistance in non-small cell lung carcinoma, respectively. Targeting of MSN to cancer cells was achieved by the conjugation of LHRH peptide on the surface of MSN via poly(ethylene glycol) spacer. The delivered anticancer drugs and siRNA preserved their specific activity leading to the cell death induction and inhibition of targeted mRNA. Suppression of cellular resistance by siRNA effectively delivered inside cancer cells and substantially enhanced the cytotoxicity of anticancer drugs. Local delivery of MSN by inhalation led to the preferential accumulation of nanoparticles in the mouse lungs, prevented the escape of MSN into the systemic circulation, and limited their accumulation in other organs. The experimental data confirm that the developed DDS satisfies the major prerequisites for effective treatment of non-small cell lung carcinoma. Therefore, the proposed cancer-targeted MSN-based system for complex delivery of drugs and siRNA has high potential in the effective treatment of lung cancer.     

Doxorubicin-loaded bacterial outer-membrane vesicles exert enhanced anti-tumor efficacy in non-small-cell lung cancer

More efficient drug delivery system and formulation with less adverse effects are needed for the clinical application of broad-spectrum antineoplastic agent doxorubicin (DOX). Here we obtained outer-membrane vesicles (OMVs), a nano-sized proteoliposomes naturally released by Gram-negative bacteria, from attenuated Klebsiella pneumonia and prepared doxorubicin-loaded O0MVs (DOX-OMV). Confocal microscopy and in vivo distribution study observed that DOX encapsulated in OMVs was efficiently transported into NSCLC A549 cells. DOX-OMV resulted in intensive cytotoxic effects and cell apoptosis in vitro as evident from MTT assay, Western blotting and flow cytometry due to the rapid cellular uptake of DOX. In A549 tumor-bearing BALB/c nude mice, DOX-OMV presented a substantial tumor growth inhibition with favorable tolerability and pharmacokinetic profile, and TUNEL assay and H&E staining displayed extensive apoptotic cells and necrosis in tumor tissues. More importantly, OMVs’ appropriate immunogenicity enabled the recruitment of macrophages in tumor microenvironment which might synergize with their cargo DOX in vivo. Our results suggest that OMVs can not only function as biological nanocarriers for chemotherapeutic agents but also elicit suitable immune responses, thus having a great potential for the tumor chemoimmunotherapy.     

Star polyester-based folate acid-targeting nanoparticles for doxorubicin and curcumin co-delivery to combat multidrug-resistant breast cancer

Chemotherapeutic treatments are indispensable in the treatment of breast cancer. However, the emergence of multidrug-resistance, strong cell toxicity, and poor targeting selection has inhibited their clinical application. In this study, two synergistic drugs, doxorubicin (DOX) and curcumin (CUR), were co-administered to overcome multidrug resistance (MDR). Based on the characteristics of the tumor microenvironment, we developed folic acid-modified nanoparticles ((DOX + CUR)-FA-NPs) based on a star-shaped polyester (FA-TRI-CL) to enhance the tumor targeting selectivity and drug loading (DL) capacity. The (DOX + CUR)-FA-NPs displayed a characteristic spheroid morphology with an ideal diameter (186.52 nm), polydispersity index (0.024), zeta potential (–18.87 mV), and good entrapment efficiency (97.64%/78.13%, DOX/CUR) and DL (20.27%/11.29%, DOX/CUR) values. In vitro pharmacokinetic and pharmacodynamic experiments demonstrated that the (DOX + CUR)-FA-NPs were gradually released, and they displayed the highest cell apoptosis and cellular uptake in MCF-7/ADR cells. Additionally, in vivo results illustrated that (DOX + CUR)-FA-NPs not only displayed significant tumor targeting and anticancer efficacy, but also induced less pathological damage to the normal tissue. In summary, co-administered DOX and CUR appeared to reverse MDR, and this targeted combinational nanoscale delivery system could thus be a promising carrier for tumor therapies in the future.     

Gp350-anchored extracellular vesicles: promising vehicles for delivering therapeutic drugs of B cell malignancies

Although chimeric antigen receptor-modified(CAR) T cell therapy has been successfully applied in the treatment of acute B lymphocytic leukemia, its effect on Burkitt lymphoma(BL) and chronic B lymphocytic leukemia(B-CLL) is unsatisfactory. Moreover, fatal side effects greatly impede CAR T cell application. Extracellular vesicles(EVs) are excellent carriers of therapeutic agents. Nevertheless, EVs mainly accumulate in the liver when administered without modification. As an envelope glycoprotein o...     

Absolute sizing and label-free identification of extracellular vesicles by flow cytometry

Blood contains extracellular vesicles (EVs), which are biological nanoparticles with clinical applications. In blood plasma, EVs are outnumbered by similar-sized lipoprotein particles (LPs), leading to controversial data such as non-specific binding of antibodies to LPs. Flow cytometry is a clinically applicable technique to characterize single EVs in body fluids. However, flow cytometry data have arbitrary units, impeding standardization, data comparison, and data interpretation, such as differentiation between EVs and LPs. Here we present a new method, named flow cytometry scatter ratio (Flow-SR), to relate the ambiguous light scattering signals of flow cytometry to the diameter and refractive index (RI) of single nanoparticles between 200-500 nm in diameter. Flow-SR enables label-free differentiation between EVs and LPs and improves data interpretation and comparison. Because Flow-SR is easy to implement, widely applicable, and more accurate and faster than existing techniques to size nanoparticles in suspension, Flow-SR has numerous applications in nanomedicine.     

Combination prostate cancer therapy: Prostate-specific membranes antigen targeted,pH-sensitive nanoparticles loaded with doxorubicin and tanshinone

Prostate cancer is the second most frequently diagnosed cancer in the men population. Combination anticancer therapy using doxorubicin (DOX) and another extract of traditional Chinese medicine is one nano-sized drug delivery system promising to generate synergistic anticancer effects, maximize the treatment effect, and overcome multi-drug resistance. The purpose of this study is to construct a drug delivery system for the co-delivery of DOX and tanshinones (TAN). Lipid nanoparticles loaded with DOX and TAN (N-DOX/TAN) were prepared by emulsification and solvent-diffusion method. PSMA targeted nanoparticles loaded with DOX and TAN (P-N-DOX/TAN) were synthesized by conjugating a PSMA targeted ligand to N-DOX/TAN. We evaluate the performance of this system in vitro and in vivo. P-N-DOX/TAN has a size of 139.7 ± 4.1 nm and a zeta potential of 11.2 ± 1.6 mV. The drug release of DOX and TAN from P-N-DOX/TAN was much faster than that of N-DOX/TAN. N-DOX/TAN presented more inhibition effect on tumor growth than N-DOX and N-TAN, which is consistent with the synergistic results and successfully highlighting the advantages of combing the DOX and TAN in one system. P-N-DOX/TAN achieved higher uptake by LNCaP cells (58.9 ± 1.9%), highest tumor tissue distribution, and the most significant tumor inhibition efficiency. The novel nanomedicine offers great promise for the dual drug delivery to prostate cancer cells, showing the potential of synergistic combination therapy for prostate cancer.     

Phase I clinical trial of doxorubicin and iproplatin combination chemotherapy in patients with breast cancer

Summary Forty-eight patients with advanced breast cancer were treated in a disease-specific phase I trial of doxorubicin and iproplatin combination chemotherapy. The doses of doxorubicin ranged between 30 and 50 mg/m₂, and the doses of iproplatin ranged between 150 and 250 mg/m₂. Myelosuppression was observed at all levels, but was dose-limiting at the highest level. In addition, nausea, diarrhea and malaise were prominent toxicities. Neither cardiac nor renal toxicity was encountered. Nine of 26 (35%) of previously untreated patients, and 5 of 22 (23%) previously treated patients demonstrated partial or complete responses. Although this combination possesses therapeutic activity, given its toxicities, further evaluation of doxorubicin in combination with iproplatin is not recommended.     

脂质体多柔比星治疗恶性肿瘤临床疗效及安全性的系统评价

目的 评价脂质体多柔比星（脂质体阿霉素）治疗恶性肿瘤的临床疗效及安全性。方法 计算机检索中国学术文献总库（CNKI）、万方数字化期刊库、中国生物医学文献数据库（CBM）、维普中文科技期刊数据库（VIP）,PubMed数据库、Embase数据库和Cochrane Library数据库,脂质体多柔比星治疗恶性肿瘤的中英文随机对照试验,检索年限从建库至2017年1月。采用RevMan 5.3软件对各效应指标进行Meta分析。结果 纳入23篇RCT文献,计5 546名恶性肿瘤患者。Meta分析结果显示：治疗前后临床疗效I²=63%、OR=1.14[1.02,1.27],P=0.002;两组化疗后心脏毒性不良事件I²=0%,OR=0.18[0.10,0.33],P<0.000 01;脱发不良事件I²=77%,OR=0.24[0.17,0.35],P<0.000 01;神经毒性不良事件I²=46%,OR=0.65[0.42,0.99],P=0.05。结论 脂质体多柔比星治疗恶性肿瘤的临床疗效优于其他化疗方案,且毒副作用低,尤其在改善心脏毒性、脱发等不良事件方面明显优于其他化疗方案。     

Population pharmacokinetics of doxorubicin, etoposide and ifosfamide in small cell lung cancer patients: results of a multicentre study

AIMS: To determine the population pharmacokinetic (PK) parameters of doxorubicin (Dox), etoposide (Eto) and ifosfamide (Ifo) in small cell lung cancer (SCLC) patients, to assess the potential relationship between those parameters and to estimate the impact of individual morphological and biological covariates on patients' PK parameters. METHODS: Twenty-four patients with either SCLC limited to the thorax or extensive SCLC entered the study. All but one received at least two 3 day courses of the standard AVI (Dox 50 mg m-2 day 1, Eto 120 mg m-2 day 1,2,3, Ifo 2000 mg m-2 day 1,2) regimen. Individual blood samples were collected during each course and data on 47 courses were available. Data were analysed with the NONMEM program. Dox, Eto and Ifo plasma concentrations were studied with multicompartment (3, 2 and 2, respectively) models. Inter-individual and interoccasion (course-to-course) variabilities were estimated. The influence of individual covariates (age, sex, stage of the disease, weight, height, body-surface area, serum creatinine, total protein, LDH, ASAT, ALAT, alkaline phosphatase, gamma-GT, bilirubin) on PK parameters was also assessed. Correlations between individual PK parameters of Dox, Eto and Ifo were explored by using Pearson's correlation coefficient. RESULTS: Multiple data were available for each patient. Dox clearance (CL) and volume of distribution (Vd) were 32.0 l h-1 and 9.3 l (Inter-individual variability: 17.2% and 19.2%). Eto CL (l h-1) and Vd were, respectively, 3.34-0.0083* serum creatinine (micromol l-1) and 6.38 l (interindividual variability: 15.6% and 18.7%). Ifo CL and Vd at day 1 were 5.6 l h-1 and 26.0 l (interindividual variability: 10.1% and 17.2%, respectively). Estimation of course-to-course variability improved the precision of PK models in some cases. No correlation was observed between the respective PK parameters of each drug. Of individual covariates tested, only serum creatinine correlated with Eto CL (r = -0.37, P < 0.001). Self-induction of the metabolism of Ifo was apparent (mean CL increase from day 1 to day 2 : 42%) and individually correlated with the CL value at day 1 (r = -0.61, P < 0.001). CONCLUSIONS: Assessment of potential relationships between individual systemic exposure of chemotherapy and therapeutic endpoints (tumour response, toxicity and survival) will be required to adjust drugs dosages based on individual PK parameters rather than questionable body-surface area. However, all three drugs in the AVI regimen should be monitored simultaneously.     

New idea to promote the clinical applications of stem cells or their extracellular vesicles in central nervous system disorders: Combining with intranasal delivery

The clinical translation of stem cells and their extracellular vesicles (EVs)-based therapy for central nervous system (CNS) diseases is booming. Nevertheless, the insufficient CNS delivery and retention together with the invasiveness of current administration routes prevent stem cells or EVs from fully exerting their clinical therapeutic potential. Intranasal (IN) delivery is a possible strategy to solve problems as IN route could circumvent the brain‒blood barrier non-invasively and fit repeated dosage regimens. Herein, we gave an overview of studies and clinical trials involved with IN route and discussed the possibility of employing IN delivery to solve problems in stem cells or EVs-based therapy. We reviewed relevant researches that combining stem cells or EVs-based therapy with IN administration and analyzed benefits brought by IN route. Finally, we proposed possible suggestions to facilitate the development of IN delivery of stem cells or EVs.Key words: Stem cells, Extracellular vesicles, Central nervous system disorders, Intranasal, Clinical translation, Stroke, Neurodegenerative disease, Glioma