An Intelligent Vascular Disrupting Dendritic Nanodevice Incorporating Copper Sulfide Nanoparticles for Immune Modulation-Mediated Combination Tumor Therapy

Development of intelligent nanoplatforms that can simultaneously target multiple factors associated with tumor growth and metastasis remains an extreme challenge. Here, an intelligent dendritic nanodevice incorporating both copper sulfide nanoparticles (CuS NPs) and 5,6-dimethylxanthenone-4-acetic acid (DMXAA, a vascular disrupting agent) within the dendrimer internal cavities and surface modified with a targeting agent LyP-1 peptide is reported. The resulting generation 5 (G5) dendrimer-based nanodevice, known as G5-PEG-LyP-1-CuS-DMXAA NPs (GLCD NPs), possess good colloidal stability, pH-sensitive drug release kinetics, and high photothermal conversion efficiency (59.3%). These functional GLCD NPs exert a LyP-1-targeted killing effect on breast tumors by combining CuS-mediated photothermal therapy (PTT) and DMXAA-induced vascular disruption, while also triggering antitumor immune responses through PTT-induced immunogenic cell death and DMXAA-mediated immune regulation via M1 polarization of tumor-associated macrophages and dendritic cell maturation. In addition, with the LyP-1-mediated proapoptotic activity, the GLCD NPs can specifically kill tumor lymphatic endothelial cells. The simultaneous disruption of tumor blood vessels and lymphatic vessels cuts off the two main pathways of tumor metastasis, which plays a two-pronged role in inhibiting lung metastasis of the breast cancer model. Thus, the developed GLCD NPs represent an advanced intelligent nanoformulation for immune modulation-mediated combination tumor therapy with potential for clinical translations.     

Multiplexed NIR-II Probes for Lymph Node-Invaded Cancer Detection and Imaging-Guided Surgery

Tumor-lymph node (LN) metastasis is the dominant prognostic factor for tumor staging and therapeutic decision-making. However, concurrently visualizing metastasis and performing imaging-guided lymph node surgery is challenging. Here, a multiplexed-near-infrared-II (NIR-II) in vivo imaging system using nonoverlapping NIR-II probes with markedly suppressed photon scattering and zero-autofluorescence is reported, which enables visualization of the metastatic tumor and the tumor metastatic proximal LNs resection. A bright and tumor-seeking donor–acceptor–donor (D-A-D) dye, IR-FD, is screened for primary/metastatic tumor imaging in the NIR-IIa (1100–1300 nm) window. This optimized D-A-D dye exhibits greatly improved quantum yield of organic D-A-D fluorophores in aqueous solutions (≈6.0%) and good in vivo performance. Ultrabright PbS/CdS core/shell quantum dots (QDs) with dense polymer coating are used to visualize cancer-invaded sentinel LNs in the NIR-IIb (>1500 nm) window. Compared to clinically used indocyanine green, the QDs show superior brightness and photostability (no obvious bleaching even after continuous laser irradiation for 5 h); thus, only a picomolar dose is required for sentinel LNs detection. This combination of dual-NIR-II image-guided surgery can be performed under bright light, adding to its convenience and appeal in clinical use.     

In Situ Tumor Vaccine for Lymph Nodes Delivery and Cancer Therapy Based on Small Size Nanoadjuvant

Tumor vaccine is a promising cancer treatment modality, however, the convenient antigens loading in vivo and efficient delivery of vaccines to lymph nodes (LNs) still remain a formidable challenge. Herein, an in situ nanovaccine strategy targeting LNs to induce powerful antitumor immune responses by converting the primary tumor into whole-cell antigens and then delivering these antigens and nanoadjuvants simultaneously to LNs is proposed. The in situ nanovaccine is based on a hydrogel system, which loaded with doxorubicin (DOX) and nanoadjuvant CpG-P-ss-M. The gel system exhibits ROS-responsive release of DOX and CpG-P-ss-M, generating abundant in situ storage of whole-cell tumor antigens. CpG-P-ss-M adsorbs tumor antigens through the positive surface charge and achieves charge reversal, forming small-sized and negatively charged tumor vaccines in situ, which are then primed to LNs. Eventually, the tumor vaccine promotes antigens uptake by dendritic cells (DCs), maturation of DCs, and proliferation of T cells. Moreover, the vaccine combined with anti-CTLA4 antibody and losartan inhibits tumor growth by 50%, significantly increasing the percentage of splenic cytotoxic T cells (CTLs), and generating tumor-specific immune responses. Overall, the treatment effectively inhibits primary tumor growth and induces tumor-specific immune response. This study provides a scalable strategy for in situ tumor vaccination.     

Enhanced radiosensitization by liposome-encapsulated pimonidazole for anticancer effects on human melanoma cells

The nitroimidazole-related hypoxic radiosensitizer, pimonidazole (Pmz) was encapsulated in liposome composed of dipalmitoylphosphatidylcholine, cholesterol and dipalmitoylphosphatidylglycerol (molar ratio = 1:1:0.2; diameter = 112.9 nm), and the radiosensitization was evaluated in human melanoma cells HMV-II. Cell proliferation was examined by WST-8 assay after X-ray irradiation in the presence of liposomal Pmz or free-Pmz under hypoxic conditions. On 7th day after X-ray irradiation of 5 Gy, cell proliferation decreased more markedly in the administration of liposomal Pmz than free-Pmz at equivalent Pmz doses. Chromatin fragmentation or nuclear condensation was observed in liposomal Pmz-treated HMV-II cells. Radiosensitization was enhanced dose-dependently along with Pmz amounts of 250-2000 microM contained in liposomal Pmz. Intracellular uptake was more abundant for liposomal Pmz for 60-240 min than for free-Pmz. Thus liposomal Pmz has a potential to overcome radiation resistance in hypoxia, owing to enhanced intracellular uptake by melanoma cells.     

Locally Trapping the C‐C Chemokine Receptor Type 7 by Gene Delivery Nanoparticle Inhibits Lymphatic Metastasis Prior to Tumor Resection

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype. Currently, no targeted treatment is available for TNBC, and the most common clinical therapy is tumor resection, which often promotes metastasis risks. Strong evidence suggests that the lymphatic metastasis is mediated by the C‐C chemokine receptor type 7 (CCR7)/C‐C motif chemokine ligand 21 crosstalk between tumor cells and the lymphatic system. It is hypothesized that CCR7 is a key immune modulator in the tumor microenvironment and the local blockade of CCR7 could effectively inhibit TNBC lymphatic metastasis. Accordingly, a plasmid encoding an antagonistic CCR7 affinity protein‐CCR7 trap is delivered by tumor targeting nanoparticles in a highly metastatic 4T1 TNBC mouse model. Results show that CCR7 traps are transiently expressed, locally disrupt the signaling pathways in the tumor site, and efficiently inhibit TNBC lymphatic metastasis, without inducing immunosuppression as observed in systemic therapies using CCR7 monoclonal antibody. Significantly, upon applying CCR7 trap therapy prior to tumor resection, a 4T1 TNBC mouse model shows good prognosis without any further metastasis and relapse. In addition, CCR7 trap therapy efficiently inhibits the lymphatic metastasis in a B16F10 melanoma mouse model, indicating its great potential for various metastatic diseases treatment.     

Dissolving Microneedle Arrays for Transdermal Delivery of Amphiphilic Vaccines

Amphiphilic vaccine based on lipid‐polymer conjugates is a new type of vaccine capable of self‐delivering to the immune system. When injected subcutaneously, amphiphilic vaccines efficiently target antigen presenting cells in the lymph nodes (LNs) via a unique albumin‐mediated transport and uptake mechanism and induce potent humoral and cellular immune responses. However, whether this new type of vaccine can be administrated via a safe, convenient microneedle‐based transdermal approach remains unstudied. For such skin barrier‐disruption systems, a simple application of microneedle arrays (MNs) is desired to disrupt the stratum corneum, and for rapid and pain‐free self‐administration of vaccines into the skin, the anatomic place permeates with an intricate mesh of lymphatic vessels draining to LNs. Here the microneedle transdermal approach is combined with amphiphilic vaccines to create a simple delivery approach which efficiently traffic molecular vaccines into lymphatics and draining LNs. The rapid release of amphiphilic vaccines into epidermis upon application of dissolving MNs to the skin of mice generates potent cellular and humoral responses, comparable or superior to those elicited by traditional needle‐based immunizations. The results suggest that the amphiphilic vaccines delivered by dissolving MNs can provide a simple and safer vaccination method with enhanced vaccine efficacy.     

Intermittent Fasting Primes the Tumor Microenvironment and Improves Nanomedicine Delivery in Hepatocellular Carcinoma

Fasting has many health benefits, including reduced chemotherapy toxicity and improved efficacy. It is unclear how fasting affects the tumor microenvironment (TME) and tumor-targeted drug delivery. Here the effects of intermittent (IF) and short-term (STF) fasting are investigated on tumor growth, TME composition, and liposome delivery in allogeneic hepatocellular carcinoma (HCC) mouse models. To this end, mice are inoculated either subcutaneously or intrahepatically with Hep-55.1C cells and subjected to IF for 24 d or to STF for 1 d. IF but not STF significantly slows down tumor growth. IF increases tumor vascularization and decreases collagen density, resulting in improved liposome delivery. In vitro, fasting furthermore promotes the tumor cell uptake of liposomes. These results demonstrate that IF shapes the TME in HCC towards enhanced drug delivery. Finally, when combining IF with liposomal doxorubicin treatment, the antitumor efficacy of nanochemotherapy is found to be increased, while systemic side effects are reduced. Altogether, these findings exemplify that the beneficial effects of fasting on anticancer therapy outcomes go beyond modulating metabolism at the molecular level.     

Phospholipid‐Free Small Unilamellar Vesicles for Drug Targeting to Cells in the Liver

It is reported that cholesterol (Chol) and TWEEN 80 at a molar ratio of 5:1 can form small unilamellar vesicles (SUVs) using a staggered herringbone micromixer. These phospholipid‐free SUVs (PFSUVs) can be actively loaded with a model drug for targeting hepatocytes via the endogenous apolipoprotein mechanism. PFSUVs particles with compositions of Chol:TWEEN 80 ranging between 1.5:1 and 5:1 (mol/mol) can be produced with a mean diameter of ≈80 nm, but only the high‐Chol formulations (3:1 and 5:1) can retain a transmembrane gradient of ammonium sulfate for active loading of doxorubicin (DOX). Under cryo‐transmission electron microscopy, PFSUVs‐DOX displays a unilamellar bilayer structure with DOX molecules forming spindle‐shape aggregates inside the aqueous core. Relative to PEGylated liposomal doxorubicin (PLD) that exhibits little interaction with cells in various conditions, the cellular uptake of PFSUVs‐DOX is dependent on the presence of serum and enhanced with an increased concentration of apolipoproteins. After intravenous injection, the vast majority of PFSUVs‐DOX accumulates in the liver and DOX is detected in all liver cells (predominantly the hepatocytes), while PLD is captured only by the sinusoidal cells (i.e., macrophages). This report discloses an innovative lipid bilayer vesicle for highly efficient and selective hepatocyte targeting.     

Continuous release of interleukin-2 from liposomal IL-2 (mixture of interleukin-2 and liposomes) after subcutaneous administration to mice

Recombinant interleukin-2 (IL-2) was strongly and almost completely adsorbed onto small and hydrophobic liposomes by simple mixing under optimal conditions (liposome: DSPC-DSPG; molar ratio, 10:1; 30-50 nm in size, ratio of IL-2 to liposome: 4.0 JRU/nmol lipid). This liposomal IL-2 displayed better distribution after intravenous administration in mice and improved therapeutic effect against experimental M5076 metastases, as reported previously.[1] In this study, the elimination of IL-2 from the dosing area was investigated when the liposomal IL-2 was administered to mice subcutaneously. The results suggest that the release of IL-2 from this liposome was continuous and almost complete. The mean residence time (MRT) of IL-2 in the dosing area was 11.0 ± 1.65 hr. This resulted in the 8-fold times enhancement of MRT in the systemic circulation by the presence of liposomes, and IL-2 was detected in the serum for 2 days. Using this liposomal IL-2 is expected to have the potential to decrease the number of injections and enhance the efficacy of IL-2 in immunotherapies and therapies against tumor.     

Overcoming Biological Barriers in Neuroblastoma Therapy: The Vascular Targeting Approach with Liposomal Drug Nanocarriers

Neuroblastoma is a rare pediatric cancer characterized by a wide clinical behavior and adverse outcome despite aggressive therapies. New approaches based on targeted drug delivery may improve efficacy and decrease toxicity of cancer therapy. Furthermore, nanotechnology offers additional potential developments for cancer imaging, diagnosis, and treatment. Following these lines, in the past years, innovative therapies based on the use of liposomes loaded with anticancer agents and functionalized with peptides capable of recognizing neuroblastoma cells and/or tumor‐associated endothelial cells have been developed. Studies performed in experimental orthotopic models of human neuroblastoma have shown that targeted nanocarriers can be exploited for not only decreasing the systemic toxicity of the encapsulated anticancer drugs, but also increasing their tumor homing properties, enhancing tumor vascular permeability and perfusion (and, consequently, drug penetration), inducing tumor apoptosis, inhibiting angiogenesis, and reducing tumor glucose consumption. Furthermore, peptide‐tagged liposomal formulations are proved to be more efficacious in inhibiting tumor growth and metastatic spreading of neuroblastoma than nontargeted liposomes. These findings, herein reviewed, pave the way for the design of novel targeted liposomal nanocarriers useful for multitargeting treatment of neuroblastoma.     

Simultaneous Quantification of Tumor Uptake for Targeted and Nontargeted Liposomes and Their Encapsulated Contents by ICPMS

Liposomes are intensively being developed for biomedical applications including drug and gene delivery. However, targeted liposomal delivery in cancer treatment is a very complicated multistep process. Unfavorable liposome biodistribution upon intravenous administration and membrane destabilization in blood circulation could result in only a very small fraction of cargo reaching the tumors. It would therefore be desirable to develop new quantitative strategies to track liposomal delivery systems to improve the therapeutic index and decrease systemic toxicity. Here, we developed a simple and nonradiative method to quantify the tumor uptake of targeted and nontargeted control liposomes as well as their encapsulated contents simultaneously. Specifically, four different chelated lanthanide metals were encapsulated or surface-conjugated onto tumor-targeted and nontargeted liposomes, respectively. The two liposome formulations were then injected into tumor-bearing mice simultaneously, and their tumor delivery was determined quantitatively via inductively coupled plasma mass spectroscopy (ICPMS), allowing for direct comparisons. Tumor uptake of the liposomes themselves and their encapsulated contents was consistent with targeted and nontargeted liposome formulations that were injected individually.     

Effect of intratumoral injection on the biodistribution and therapeutic potential of novel chemophor EL-modified single-walled nanotube loading doxorubicin

The purpose of this study is to evaluate in vivo efficacy and loco-regional distribution of a doxorubicin (DOX)-loaded Polyoxyl 35 Castor Oil (Cremophor EL, CrEL) noncovalent modified single-walled carbon nanotubes (SWNTs) formulation in a sarcoma tumor model after intratumoral injection. The drug loaded SWNTs were successfully prepared via physical absorption, which was confirmed by UV-vis-NIR absorbance spectra and dynamic light scattering assay. Solid tumor models were obtained by injecting mouse sarcoma 180 cells into the thighs of ICR mice. CrEL-SWNTs-DOX, CrEL-SWNTs, free DOX and saline (control) were intratumorally injected after 5 days post transplantation. The biodistribution studies demonstrated that intratumoral delivery of CrEL-SWNTs-DOX resulted in longer drug retention time in tumor, higher tumor level (27.6-fold than that of free DOX), as well as less accumulation in other solid tissues, especially in heart. Furthermore, in vivo anti-tumor activity results showed that CrEL-SWNTs-DOX could effectively suppress the tumor growth than free DOX and the control, attributing to its enhanced intratumoral DOX level. The histopathological findings revealed that the new carbon nanomaterials were a safe vehicle for topical drug delivery systems. It is concluded that this noncovalent modification of carbon nanotubes by CrEL for anticancer agents might be a promising alternative for cancer treatment.     

Characteristics and photo-responsive release property of liposome containing 7-acetoxy coumarin

A photo-responsive liposome was developed by loading 7-acetoxy coumarin (ATC) in egg phosphatidylcholine (EPC) liposome. ATC was derivetized from 7-hydroxy coumarin using sodium acetate. The ATC-loaded liposomes were prepared by suspending the dry mixture film of ATC/EPC in distilled water and sonicating the suspension. When the ATC to EPC ratio was less than 1:8, homogeneous liposomal suspensions were obtained without any precipitate. On 1H NMR spectra, the unsaturated chains of liposomal EPCs were somewhat deteriorated by a subsequent irradiation, the irradiation of lambda = 365 nm and then the irradiation of lambda = 254 nm. On TEM photos, the size of liposomes incorporating ATC markedly increased by the subsequent irradiation. Photo-dimerization (under lambda = 365 nm) and de-dimerization (under lambda = 254 nm) of ATC took a place even in EPC liposomes. Upon the irradiation of lambda = 254 nm, liposome containing ATC dimers exhibited an enhanced release of 5(6)-carboxyfluorescein compared with liposome containing ATC monomer and liposome free of ATC. The de-dimerization of ATC dimers is believed to fluidize the liposomal membrane and promote the release.     

Self‐Assembly of an Amphiphilic Janus Camptothecin–Floxuridine Conjugate into Liposome‐Like Nanocapsules for More Efficacious Combination Chemotherapy in Cancer

The combination of camptothecin (CPT) and fluoropyrimidine derivatives acts synergistically at a 1:1 molar ratio. Practically, the greatest challenge is the development of a single liposomal formulation that can both encapsulate and maintain this drug combination at an exact 1:1 ratio to achieve coordinated pharmacokinetics. Consequently, a new type of liposome‐like nanocapsule (NC) is developed from a highly symmetric Janus camptothecin–floxuridine conjugate (JCFC) amphiphile, which is synthesized by coupling two hydrophobic CPT molecules and two hydrophilic floxuridine (FUDR) molecules to multivalent pentaerythritol via a hydrolyzable ester linkage. JCFC NCs possess remarkably high drug‐loading contents, and no premature release because of the highly stable co‐delivery of the drug combination without the need for any carrier. It is shown that JCFC NCs consistently provide synergy and avoid antagonism in a broad panel of tumor cell lines. In vivo delivery of JCFC NCs leads to longer blood retention half‐life, higher tumorous accumulation and cellular uptake of drugs, and greatly enhanced efficacy in murine tumor models compared to CPT, FUDR, and CPT + FUDR. This liposomal strategy can be extended to other hydrophilic and hydrophobic anticancer drugs that are coupled to pentaerythritol to self‐assemble into nanocapsules for drug self‐delivery, pointing to potential clinical translation in near future.     

In Vivo Environment‐Adaptive Nanocomplex with Tumor Cell–Specific Cytotoxicity Enhances T Cells Infiltration and Improves Cancer Therapy

Drug delivery strategies possessing selectivity for cancer cells are eagerly needed in therapy of metastatic breast cancer. In this study, the chemotherapeutic agent, docetaxel (DTX), is conjugated onto heparan sulfate (HS). Aspirin (ASP), which has the activity of anti‐metastasis and enhancing T cells infiltration in tumors, is encapsulated into the HS‐DTX micelle. Then the cationic polyethyleneimine (PEI)‐polyethylene glycol (PEG) copolymer binds to HS via electrostatic force, forming the ASP‐loaded HS‐DTX micelle (AHD)/PEI‐PEG nanocomplex (PAHD). PAHD displays long circulation behavior in blood due to the PEG shell. Under the tumor microenvironment with weakly acidic pH, PEI‐PEG separates from AHD, and the free cationic PEI‐PEG facilitates the cellular uptake of AHD by increasing permeability of cell membranes. Then the overexpressed heparanase degrades HS, releasing ASP and DTX. PAHD shows specific toxicity toward tumor cells but not normal cells, with advanced activity of inhibiting tumor growth and lung metastasis in 4T1 tumor‐bearing mice. The number of CD8⁺ T cells in tumor tissues is also increased. Therefore, PAHD can become an efficient drug delivery system for breast cancer treatment.     

近红外荧光磁性复合载药脂质体的制备及应用

拟建立以近红外荧光磁性复合脂质体(NFMSLs)为模型药物载体、盐酸多柔比星(DOX)为包封药物的药物输送系统,研究了近红外荧光磁性载药复合脂质体(DOX-NFMSLs)的制备、性质及初步应用.采用共沉淀法制备FeO4磁流体,CdTe掺杂Se制备近红外量子点CdSeTe,薄膜分散法制备DOX-NFMSLs.用DOX荧光分光光度法测定DOX-NFMSLs的包封率和体外药物释放率;用DOX-NFMSLs与HepG2肝癌细胞共孵育来进行细胞成像和细胞毒性实验.结果表明,近红外CdSeTe量子点粒径约为5nm,闪锌矿结构,发射波长824 nm.磷脂与胆固醇质量比为8∶1,药脂比为1∶20的DOX-NFMSLs平均粒径为252.9 nm,Zeta电位为-48.6 mV,理想释放药物温度为41℃,平均包封率为(74.84±0.89)％.DOX-NFMSLs对HepG2肝癌细胞有一定的抗癌效果.得到了具有良好磁响应、释药温度T=41℃、可近红外成像的载药脂质体.     

Effect of intratumoral injection on the biodistribution and therapeutic potential of novel chemophor EL-modified single-walled nanotube loading doxorubicin

The purpose of this study is to evaluate in vivo efficacy and loco-regional distribution of a doxorubicin (DOX)-loaded Polyoxyl 35 Castor Oil (Cremophor EL, CrEL) noncovalent modified single-walled carbon nanotubes (SWNTs) formulation in a sarcoma tumor model after intratumoral injection. The drug loaded SWNTs were successfully prepared via physical absorption, which was confirmed by UV-vis-NIR absorbance spectra and dynamic light scattering assay. Solid tumor models were obtained by injecting mouse sarcoma 180 cells into the thighs of ICR mice. CrEL-SWNTs-DOX, CrEL-SWNTs, free DOX and saline (control) were intratumorally injected after 5 days post transplantation. The biodistribution studies demonstrated that intratumoral delivery of CrEL-SWNTs-DOX resulted in longer drug retention time in tumor, higher tumor level (27.6-fold than that of free DOX), as well as less accumulation in other solid tissues, especially in heart. Furthermore, in vivo anti-tumor activity results showed that CrEL-SWNTs-DOX could effectively suppress the tumor growth than free DOX and the control, attributing to its enhanced intratumoral DOX level. The histopathological findings revealed that the new carbon nanomaterials were a safe vehicle for topical drug delivery systems. It is concluded that this noncovalent modification of carbon nanotubes by CrEL for anticancer agents might be a promising alternative for cancer treatment.     

Pulmonary Codelivery of Doxorubicin and siRNA by pH‐Sensitive Nanoparticles for Therapy of Metastatic Lung Cancer

A pulmonary codelivery system that can simultaneously deliver doxorubicin (DOX) and Bcl2 siRNA to the lungs provides a promising local treatment strategy for lung cancers. In this study, DOX is conjugated onto polyethylenimine (PEI) by using cis‐aconitic anhydride (CA, a pH‐sensitive linker) to obtain PEI‐CA‐DOX conjugates. The PEI‐CA‐DOX/siRNA complex nanoparticles are formed spontaneously via electrostatic interaction between cationic PEI‐CA‐DOX and anionic siRNA. The drug release experiment shows that DOX releases faster at acidic pH than at pH 7.4. Moreover, PEI‐CA‐DOX/Bcl2 siRNA complex nanoparticles show higher cytotoxicity and apoptosis induction in B16F10 cells than those treated with either DOX or Bcl2 siRNA alone. When the codelivery systems are directly sprayed into the lungs of B16F10 melanoma‐bearing mice, the PEI‐CA‐DOX/Bcl2 siRNA complex nanoparticles exhibit enhanced antitumor efficacy compared with the single delivery of DOX or Bcl2 siRNA. Compared with systemic delivery, most drug and siRNA show a long‐term retention in the lungs via pulmonary delivery, and a considerable number of the drug and siRNA accumulate in tumor tissues of lungs, but rarely in normal lung tissues. The PEI‐CA‐DOX/Bcl2 siRNA complex nanoparticles are promising for the treatment of metastatic lung cancer by pulmonary delivery with low side effects on the normal tissues.