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In this study, the strategy of transient generation of holes in the liposome surface has been shown to enable safe encapsulation of a high-molecular weight antibody (rituximab, Mw∼140 kDa) within liposomes. These transient holes generated using our magnetoporation method allowed rituximab to safely enter the liposomes, and then the holes were plugged using hyaluronic acid grafted with 3-diethylaminopropylamine (DEAP). In the tumor microenvironment, the resulting liposomal rituximab was destabilized because of the ionization of the DEAP moiety at the acidic pH 6.5, resulting in extensive release of rituximab. Consequently, the rituximab released from the liposomes accumulated at high levels in tumors and bound to the CD20 receptors overexpressed on Burkitt lymphomaRamos cells. This event led to significant enhancement in tumor cell ablation through rituximab-mediated complement-dependent cytotoxicity and Bcl-2 signaling inhibition-induced cell apoptosis.

Exosomes have been widely demonstrated as an effective anticancer therapeutic moiety. However, their clinical translation has been limited by the requirement of prohibitively high therapeutic doses due to their lack of specificity in delivery and, consequently, short systemic half-life. To overcome these challenges, we engineered a platform for modifying exosomes with an active targeting modality composed of membrane Anchor (BODIPY)-Spacer (PEG)-targeting Ligands (cyclic RGD peptide) (ASL). Herein, we show that the intramembrane incorporation of a trackable, targeting system renders ASL exosomes (AExs) a modular platform. AExs significantly overcome challenges associated with exosome modification, including potential damage for functionalization, or destabilizing interactions between dyes and drugs. ASL-modification not only enhanced stability in imparting active targeting but also introduced a built-in bioimaging modality. Our studies show that AExs target B16F10 melanoma tumor sites by the specific interaction of cyclic RGD and integrin. Doxorubicin encapsulated AExs (dAExs) significantly inhibited the growth of melanoma in vitroand in vivo. Thus, we conclude that ASL-modification allows exosomes to be transformed into a novel therapeutic vehicle uniquely integrating in vivotracking and robust targeting with drug delivery. We anticipate that the therapeutic, targeting, and diagnostic modularity provided by ASL will potentiate translational applications of exosome-based vehicles beyond anticancer therapy.

Because of the complexity of cancer, an ideal anticancer strategy is better to target both cancer cells and the tumor microenvironment. In this study, for the first time, we demonstrated that zinc oxide nanoparticles (ZnO NPs) were able to target multiple cell types of cancer, including cancer cells, cancer stem cells (CSCs), and macrophages, and simultaneously perform several key functions, including inhibition of cancer proliferation, sensitization of drug-resistant cancer, prevention of cancer recurrence and metastasis, and resuscitation of cancer immunosurveillance. As a nanocarrier, the chemotherapy drug, doxorubicin (Dox), could be loaded to ZnO NPs and the Dox-loaded ZnO NPs (ZnO/Dox) possessed excellent physicochemical and pH-responsive drug release properties. ZnO/Dox could be effectively internalized by both drug-sensitive and multidrug resistant (MDR) cancer cells and penetrate more efficiently through three-dimensional (3D) cancer cell spheroids compared with free Dox. As a cytotoxic agent, ZnO NPs were more efficient to kill MDR cancer cells. Interestingly, neither ZnO nor Dox showed high cytotoxicity in the 3D cancer cell spheroids, whereas ZnO/Dox showed remarkable synergistic anticancer effects. More importantly, we demonstrated that ZnO NPs could effectively downregulate CD44, a key CSC surface marker, and decrease the stemness of CSCs, leading to the sensitization of the Dox treatment, inhibition of the cancer cell adhesion and migration, and prevention of the tumor (3D cancer cell spheroid) formation. As an immunomodulator, ZnO NPs could protect macrophages from the Dox-induced toxicity and boost the Dox-induced macrophage polarization toward an M1-like phenotype. The macrophage-conditioned medium could promote the cancer cell apoptosis in both cancer cell monolayers and 3D spheroids. The findings in this study indicated that ZnO NPs were a multifunctional and multitarget nanocarrier and nanomedicine that would have more profound effects on cancer treatment.

To improve tumor targetability and drug efficacy and decrease drug resistance of dasatinib (DSB), the multifunctional micellar nanoparticles that combined the matrix metalloproteinase 2 (MMP2)-sensitive tumor (site) targeting with folate receptor-mediated tumor (cell) targeting were developed. Two major functional polymers, polyethylene glycol (5000 Da)–MMP2-sensitive peptide–phosphoethanolamine (PEG5k-pp-PE) and folic acid–polyethylene glycol (2000 Da)–phosphoethanolamine (FA-PEG2k-PE), were synthesized to construct the dual-targeted micellar nanoparticles (MMP/FR micelles). In the absence of MMP2, the FA was shielded by PEG5k and the MMP/FR micelles showed low bioactivity. In the presence of MMP2, the nanoparticulate structure, stability, and cargo release profile of the MMP/FR micelles were not significantly affected, however, the MMP2-mediated PEG5k deshielding and FA exposure remarkably increased the cellular uptake and anticancer activity of the micelles in the MMP2 and FR expressing (MMP2+/FR+) cells, including multidrug resistant (MDR) cancer cells, rather than the MMP2– and FR– cells. In the 3D MDR tumor spheroids, the significant MMP2-dependent tissue penetration, uptake and cytotoxicity of the MMP/FR micelles were also observed. Furthermore, in the in vivo biodistribution study, the MMP2 and FR dual targeting strategy could significantly prolong the systemic circulation, decrease the nonspecific distribution in nontumor tissues, and increase the tumor accumulation of the polymeric micelles in a melanoma xenograft mouse model. The MMP2-sensitive FR-targeted micelles might have great potential as a tumor-targeted platform for delivery of molecular targeted therapeutics.

In this study, we described an “all-in-one” polymer–lipid conjugate (PEG2k-ppTAT-PEG1k-PE) that could self-assemble to matrix metalloproteinase 2 (MMP2)-sensitive multifunctional micelles. The assembled micelles had several key features, including a protective long chain poly(ethylene glycol) (PEG2k) (the outer shell), an MMP2-sensitive peptide linker (pp) (the tumor-targeting middle layer), a trans-activating transcriptional activator (TAT) peptide (the cell-penetrating middle layer), and a stable PEG1k-PE micelle for drug loading (the inner core). In the absence of MMP2, the PEG2k-ppTAT-PEG1k-PE micelles were intact and showed low bioactivity due to the surface-anchored PEG2k, whereas in the presence of MMP2, the pp was cleaved, resulting in the PEG2k deshielding and exposure of the previously hidden TAT for enhanced intracellular drug delivery. Even if completely cleaved by MMP2, the remaining PEG1k-PE micelles were stable and the micelles’ particle size and drug release were not significantly influenced. The paclitaxel (PTX)-loaded PEG2k-ppTAT-PEG1k-PE micelles showed significant MMP2-dependent cellular uptake, tumor penetration, and anticancer activity in various cancer cells and three-dimensional multicellular spheroids. Because of the enhanced intracellular drug accumulation, these multifunctional micelles were able to sensitize the drug-resistant cancer cells and their spheroids to PTX treatments. Furthermore, in vivo tumor uptake and retention data indicated that the PEG2k-ppTAT-PEG1k-PE micelles could dramatically increase the residence time of their payloads in the tumor.

The novel self-assembling bottlebrush polyethylene glycol-polynorbornene-thiocresol block copolymers (PEG-PNB-TC) were synthesized by the ring opening metathesis polymerization (ROMP), followed by functionalization of the polymer backbone via the thio-bromo “click” postpolymerization strategy. The PEG-PNB-TC copolymers could easily self-assemble into the nanoscale core–shell polymeric micelles. The hydrophobic anticancer drugs, such as paclitaxel (PTX), could be loaded into their hydrophobic core to form a stable drug-loaded micelle with a superior drug loading capacity of up to ∼35% (w/w). The sustained drug release behavior of the PEG-PNB-TC micelles was observed under a simulated “sink condition”. Compared with commercial PTX formulation (Taxol), the PTX-loaded PEG-PNB-TC micelles showed the enhanced in vitro cellular uptake and comparable cytotoxicity in the drug-sensitive cancer cells, while the copolymers were much safer than Cremophor EL, the surfactant used in Taxol. Furthermore, curcumin (CUR), a natural chemotherapy drug sensitizer, was successfully coloaded with PTX into the PEG-PNB-TC micelles. High drug loading capacity of the PEG-PNB-TC micelles allowed for easy adjustment of drug doses and the ratio of the coloaded drugs. The combination of PTX and CUR showed synergistic anticancer effect in both the drug mixture and drug coloaded micelles at high CUR/PTX ratio, while low CRU/PTX ratio only exhibited additive effects. The combinatorial effects remarkably circumvented the PTX resistance in the multidrug resistant (MDR) cancer cells. Due to the easy polymerization and functionalization, excellent self-assembly capability, high drug loading capability, and great stability, the PEG-PNB-TC copolymers might be a promising nanomaterial for delivery of the hydrophobic anticancer drugs, especially for combination drug therapy.

Since current treatment options applicable to cancers are still confined to chemotherapeutics followed by surgical debulking, cancer disease is a second leading cause of death because of chemotherapeutics of non-specific biodistribution. Image-guided drug delivery system (IGDDS) is a real-time noninvasive imaging assessment of therapeutic response that has the strong potential to control or suppress cancer because imaging property offer the quantification of nanomedicine at the intended disease site, thus the potential assurance of adequate treatment and elimination of undesirable delay leading to poor clinical outcomes. Also, combining pH-sensitive advantages of nanoparticle with theranostic property will have high chance to overcome cancer. There have been intensive researches to develop innovative IGDDS by using nanoparticles including organic or inorganic theranostic (therapy + diagnostic) systems including liposomes, polymeric micelles, dendrimers, carbon nanotubes, gold nanoparticles, iron oxide nanoparticles, quantum dots, and silica nanoparticles. In this review, we will introduce various examples of each system and will discuss their theranostic applications.

ABSTRACTSeveral angiogenesis inhibitors targeting the vascular endothelial growth factor (VEGF) signaling pathway have been approved for cancer treatment. However, VEGF inhibitors alone were shown to promote tumor invasion and metastasis by increasing intratumoral hypoxia in some preclinical and clinical studies. Emerging reports suggest that Delta-like ligand 4 (Dll4) is a promising target of angiogenesis inhibition to augment the effects of VEGF inhibitors. To evaluate the effects of simultaneous blockade against VEGF and Dll4, we developed a bispecific antibody, HD105, targeting VEGF and Dll4. The HD105 bispecific antibody, which is composed of an anti-VEGF antibody (bevacizumab-similar) backbone C-terminally linked with a Dll4-targeting single-chain variable fragment, showed potent binding affinities against VEGF (KD: 1.3 nM) and Dll4 (KD: 30 nM). In addition, the HD105 bispecific antibody competitively inhibited the binding of ligands to their receptors, i.e., VEGF to VEGFR2 (EC50: 2.84 ± 0.41 nM) and Dll4 to Notch1 (EC50: 1.14 ± 0.06 nM). Using in vitro cell-based assays, we found that HD105 effectively blocked both the VEGF/VEGFR2 and Dll4/Notch1 signaling pathways in endothelial cells, resulting in a conspicuous inhibition of endothelial cell proliferation and sprouting. HD105 also suppressed Dll4-induced Notch1-dependent activation of the luciferase gene. In vivo xenograft studies demonstrated that HD105 more efficiently inhibited the tumor progression of human A549 lung and SCH gastric cancers than an anti-VEGF antibody or anti-Dll4 antibody alone. In conclusion, HD105 may be a novel therapeutic bispecific antibody for cancer treatment.

Effective delivery of therapeutics to the brain is challenging. Molecular shuttles use receptors expressed on brain endothelial cells to deliver therapeutics. Antibodies targeting transferrin receptor (TfR) have been widely developed as molecular shuttles. However, the TfR-based approach raises concerns about safety and developmental burden. Here, we report insulin-like growth factor 1 receptor (IGF1R) as an ideal target for the molecular shuttle. We also describe Grabody B, an antibody against IGF1R, as a molecular shuttle. Grabody B has broad cross-species reactivity and does not interfere with IGF1R-mediated signaling. We demonstrate that administration of Grabody B-fused anti-alpha-synuclein (α-Syn) antibody induces better improvement in neuropathology and behavior in a Parkinson’s disease animal model than the therapeutic antibody alone due to its superior serum pharmacokinetics and enhanced brain exposure. The results indicate that IGF1R is an ideal shuttle target and Grabody B is a safe and efficient molecular shuttle.

The second generation of pH-sensitive micelles composed of poly(l-histidine-co-l-phenlyalanine(16 mol %))(MW:5K)-b-PEG(MW:2K) and poly(l-lactic acid)(MW:3K)-b-PEG(MW:2K)-folate (80/20 wt/wt %) was previously optimized by physicochemical and in vitro tests for both folate receptor and early endosomal pH targeting (pH ∼6.0). In this study, the therapeutic efficacy of the doxorubicin (DOX)-loaded micelles (DOX loading content: 20 wt %) was evaluated using in vivo tests. Multidrug-resistant (MDR) ovarian tumor-xenografted mouse models were employed. The skin-fold dorsal window chamber model was applied for visualization of extravasation and drug retention for the initial one hour after iv injection. Noninvasive imaging followed, providing evidence of drug accumulation in the tumor after the first hour. The biodistribution study further supported the long circulation of the drug carrier, tumor-selective accumulation and intracellular drug delivery. Comprehensive tumor growth inhibition experiments examined the collective efficacy of the pH-sensitive micelles. The micelle formulation effectively suppressed the growth of existing MDR tumors in mice for at least 50 days by three iv injections at a 3-day interval at a dose of 10 mg of DOX/kg. The body weight of the animals treated with the test micelle formulation gradually increased over the experimental time period, rather than decreasing. The micelle formulation was superior to its first generation, which targeted pH 6.8 and folate receptor.

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Notch signaling has been identified as a critical pathway in gastric cancer (GC) progression and metastasis, and inhibition of Delta-like ligand 4 (DLL4), a Notch ligand, is suggested as a potent therapeutic approach for GC. Expression of both DLL4 and vascular endothelial growth factor receptor 2 (VEGFR2) was similar in the malignant tissues of GC patients. We focused on vascular endothelial growth factor (VEGF), a known angiogenesis regulator and activator of DLL4. Here, we used ABL001, a DLL4/VEGF bispecific therapeutic antibody, and investigated its therapeutic effect in GC. Treatment with human DLL4 therapeutic antibody (anti-hDLL4) or ABL001 slightly reduced GC cell growth in monolayer culture; however, they significantly inhibited cell growth in 3D-culture, suggesting a reduction in the cancer stem cell population. Treatment with anti-hDLL4 or ABL001 also decreased GC cell migration and invasion. Moreover, the combined treatment of irinotecan with anti-hDLL4 or ABL001 showed synergistic antitumor activity. Both combination treatments further reduced cell growth in 3D-culture as well as cell invasion. Interestingly, the combination treatment of ABL001 with irinotecan synergistically reduced the GC burden in both xenograft and orthotopic mouse models. Collectively, DLL4 inhibition significantly decreased cell motility and stem-like phenotype and the combination treatment of DLL4/VEGF bispecific therapeutic antibody with irinotecan synergistically reduced the GC burden in mouse models. Our data suggest that ABL001 potentially represents a potent agent in GC therapy. Further biochemical and pre-clinical studies are needed for its application in the clinic. [BMB Reports 2020; 53(10): 533-538].