Co-delivery of doxorubicin and itraconazole by Pluronic® P123 coated liposomes to enhance the anticancer effect in breast cancers

To date, the combinational cancer therapy of anticancer and antiangiogenic agents represents a promising strategy to improve antitumor outcomes in clinics. However, combination therapy with drugs having distinct properties, such as solubility, limits the likelihood of simultaneous delivery. In our study, we aimed to develop a codelivery nanoparticulate system of hydrophilic doxorubicin (DOX) and hydrophobic itraconazole (ITZ) using liposomes coated with Pluronic® P123 (ITZ/DOX-PLip). The prepared ITZ/DOX-PLip exhibited a unimodal size distribution and high loading efficiency with sustained release profiles. Furthermore, cytotoxicity against 4T1 murine breast cancer cells and cellular uptake results revealed that the inhibitory effect of ITZ/DOX-Plip on tumor growth was superior to that of free DOX or DOX-loaded liposome (DOX-Lip), which was primarily attributed to the significantly higher intercellular DOX content. Cytotoxicity against HUVEC and wound healing tests confirmed that ITZ and ITZ formulations could inhibit the growth and migration of endothelial cells. In addition, in xenograft 4T1 bearing BALB/c mice, biodistribution experiments revealed that higher drug accumulation in tumors and decreased distribution in heart were observed for ITZ/DOX-PLip as compared to free DOX. Remarkably, ITZ/DOX-PLip significantly reduced tumor volume, tumor weight, liver metastasis and microvessel density in comparison with the same dose of ITZ injection or DOX-Lip. Overall, this Pluronic® P123 modified liposome-based codelivery system represents a promising nano-platform for combination therapy in cancers.

A Pluronic® P123 modified liposome-based co-delivery system of hydrophilic doxorubicin and hydrophobic itraconazole for enhanced anticancer effect in breast cancers.     

Biocompatible supramolecular pseudorotaxane hydrogels for controllable release of doxorubicin in ovarian cancer SKOV-3 cells

A series of injectable and biocompatible delivery DOX-loaded supramolecular hydrogels were fabricated by using presynthesized DOX-2N-β-CD, Pluronic F-127 and α-CD through host–guest interactions and cooperative multivalent hydrogen bonding interactions. The compositions and morphologies of these hydrogels were confirmed by PXRD and SEM measurements. Moreover, the Rheological measurements of these hydrogels were studied and the studies found that they showed a unique thixotropic behavior, indicting a fast self-healing property after the continuous oscillatory shear stress. Using α-CD as a capping agent, slow and sustained DOX release was observed at different pH values after 72 h. The amount of DOX released at pH 7.4 was determined to be 49.0% for hydrogel 1, whereas the releasing amount of the DOX was increased to 66.3% for hydrogel 1 during the same period at pH 5.5 (P < 0.05), indicating a higher release rate of the drug under more acidic conditions. Taking hydrogel 1 as a representative material, the toxicities of DOX and hydrogel 1 on ovarian cancer cells (SKOV-3) at different exposure durations were examined. The results revealed that hydrogel 1 was less cytotoxic than free DOX to SKOV-3 cells (P < 0.05), suggesting sustained release by these hydrogels in the presence of ovarian cancer cells. It is anticipated that this exploration can provide a new strategy for preparing drug delivery systems.

A series of injectable and biocompatible delivery DOX-loaded supramolecular hydrogels were fabricated by using presynthesized DOX-2N-β-CD, Pluronic F-127 and α-CD through host–guest interactions and cooperative multivalent hydrogen bonding interactions.     

A non-RGD-based integrin binding peptide (ATN-161) blocks breast cancer growth and metastasis in vivo

PURPOSE: Integrins are expressed by numerous tumor types including breast cancer, in which they play a crucial role in tumor growth and metastasis. In this study, we evaluated the ability of ATN-161 (Ac-PHSCN-NH2), a 5-mer capped peptide derived from the synergy region of fibronectin that binds to alpha5beta1 and alphavbeta3 in vitro, to block breast cancer growth and metastasis. EXPERIMENTAL DESIGN: MDA-MB-231 human breast cancer cells were inoculated s.c. in the right flank, or cells transfected with green fluorescent protein (MDA-MB-231-GFP) were inoculated into the left ventricle of female BALB/c nu/nu mice, resulting in the development of skeletal metastasis. Animals were treated with vehicle alone or by i.v. infusion with ATN-161 (0.05-1 mg/kg thrice a week) for 10 weeks. Tumor volume was determined at weekly intervals and tumor metastasis was evaluated by X-ray, microcomputed tomography, and histology. Tumors were harvested for histologic evaluation. RESULT: Treatment with ATN-161 caused a significant dose-dependent decrease in tumor volume and either completely blocked or caused a marked decrease in the incidence and number of skeletal as well as soft tissue metastases. This was confirmed histologically as well as radiographically using X-ray and microcomputed tomography. Treatment with ATN-161 resulted in a significant decrease in the expression of phosphorylated mitogen-activated protein kinase, microvessel density, and cell proliferation in tumors grown in vivo. CONCLUSION: These studies show that ATN-161 can block breast cancer growth and metastasis, and provides a rationale for the clinical development of ATN-161 for the treatment of breast cancer.     

The tetrameric peptide LfcinB (20–25)4 derived from bovine lactoferricin induces apoptosis in the MCF-7 breast cancer cell line

The cytotoxic effect of the tetrameric peptide LfcinB (20–25)₄ against breast cancer cell line ATCC® HTB-22™ (MCF-7) was evaluated. The tetrameric peptide exhibited a concentration-dependent cytotoxic effect against MCF-7 cancer cells. The peptide at 22 µM had the maximum cytotoxic effect against MCF-7 cancer cells, reducing their cell viability to ∼20%. The cytotoxic effect of the tetrameric peptide against MCF-7 cells was sustained for 24 hours. Furthermore, the tetrameric peptide did not exhibit a significant cytotoxic effect against the non-tumorogenic trophoblastic cell line, which confirms their selectivity for breast cancer cell lines. The MCF-7 cells treated at 12.2 µM for 1 h exhibited morphological changes characteristic of apoptosis, such as rounded forms and cellular shrinkage. Furthermore, this peptide induces severe cellular damage to MCF-7 cells, mitochondrial membrane depolarization, and increase of cytoplasmic calcium concentration. Our results suggest that it has a significant selective cytotoxic effect against MCF-7 cells, which may be mainly associated with the apoptotic pathway. This peptide, which contains the RRWQWR motif, could be considered to be a promising candidate for developing therapeutic agents for the treatment of breast cancer.

The cytotoxic effect of the tetrameric peptide LfcinB (20–25)₄ against breast cancer cell line ATCC® HTB-22™ (MCF-7) was evaluated.     

Characterization and targeting ability evaluation of cell-penetrating peptide LyP-1 modified alginate-based nanoparticles

Doxorubicin hydrochloride (DOX) shows a powerful treatment effect on breast cancer. However, for its indiscriminate distribution after systemic administration, the therapeutic response of DOX will reduce and even result in serious adverse reactions during the long-term administration. To achieve better treatment, in this study we established a non-condensing sodium alginate-based nanoparticle-encapsulated DOX (DOX/NP), the surface of which was modified with cell-penetrating peptide LyP-1 (namely LyP-1-DOX/NP) to attain active targeting towards breast cancer cells. The size of LyP-1-DOX/NP was 138.50 ± 4.65 nm, with a polydispersity index (PDI) of 0.22 ± 0.02, and the zeta potential was 18.60 ± 0.49 mV. The drug loading efficiency (DLE) for the preparation was 91.21 ± 2.01%, with an encapsulation efficiency (EE) of 12.37 ± 0.35%. The nanoparticles exhibited good stability in vitro and slower release trend compared with free DOX in PBS at pH7.4. In vitro cytopharmacodynamics showed that LyP-1-DOX/NP had an excellent anti-breast cancer effect against MDA-MB-231 cells by the MTT test. The uptake amount of LyP-1-DOX/NP by MDA-MB-231 cells was much higher than that of free DOX or unmodified DOX/NP at all time points. Further in vivo pharmacokinetics studies showed that the concentration of LyP-1-DOX/NP was higher than that of free DOX or DOX/NP both in plasma and in tumor, suggesting its favorable long circulation and enhancing targeting property. The present study provides a promising strategy for using the LyP-1 cell-penetrating peptide to modify nanoparticles for enhancing their targeting ability towards breast cancer.

Doxorubicin hydrochloride (DOX) shows a powerful treatment effect on breast cancer.     

Dual-stimuli-responsive TiOx/DOX nanodrug system for lung cancer synergistic therapy

Biological applications of nanosheets are rapidly increasing currently, which introduces new possibilities to improve the efficacy of cancer chemotherapy and radiotherapy. Herein, we designed and synthesized a novel nano-drug system, doxorubicin (DOX) loaded titanium peroxide (TiO_x) nanosheets, toward the synergistic treatment of lung cancer. The precursor of TiO₂ nanosheets with high specific surface area was synthesized by a modified hydrothermal process using the polymer P123 as a soft template to control the shape. TiO_x nanosheets were obtained by oxidizing TiO₂ nanosheets with H₂O₂. The anti-cancer drug DOX was effectively loaded on the surface of TiO_x nanosheets. Generation of reactive oxygen species, including H₂O₂, ·OH and ·O₂⁻, was promoted from TiO_x nanosheets under X-ray irradiation, which is effective for cancer radiotherapy and drug release in cancer cells. In this way, chemotherapy and radiotherapy were combined effectively for the synergistic therapy of cancers. Our results reinforce the DOX loaded TiO_x nanosheets as a pH sensitive and X-ray controlled dual-stimuli-responsive drug release system. The cytotoxicity, cellular uptake, and intracellular location of the formulations were evaluated in the A549 human non-small cell lung cancer cell line. Our results showed that TiO_x/DOX complexes exhibited a greater cytotoxicity toward A549 cells than free DOX. This work demonstrates that the therapeutic efficacy of DOX-loaded TiO_x nanosheets is strongly dependent on their loading mode and the chemotherapeutic and radiotherapy effect is improved under X-ray illumination, which provides a significant breakthrough for future applications of TiO_x as a light activated drug carrier in cancer chemotherapy and radiotherapy.

TiO_x/DOX nanosheets are synthesized and used as a novel nanodrug system, which introduces new possibilities to improve the efficacy of cancer by the synergistic therapy of chemotherapy and radiotherapy.     

Pluronic F127 self-assembled MoS2 nanocomposites as an effective glutathione responsive anticancer drug delivery system

In this study, bio-responsive polymeric MoS₂ nanocomposites were prepared for use as a drug carrier for cancer therapy. Herein, we report the synthesis and demonstrate the self-assembly of pluronic F127 (PF127) on a cystamine–glutathione–MoS₂ (CYS–GSH–MoS₂) system, which can be used for GSH-triggered drug release under biological reducing conditions. The reduction-sensitive disulfide bond containing CYS was incorporated between the amphiphilic copolymer PF127 and GSH–MoS₂ to achieve feasible drug release. Percent drug loading capacity and encapsulation efficiency were 51.3% and 56%, respectively. In addition, when the MoS₂–GSH–CYS–PF127 nanocomposite was incubated in a GSH environment, the morphology of the nanocomposite tended to change, ultimately leading to drug release. The drug-loaded PF127–CYS–GSH–MoS₂ polymeric nanocomposites efficiently released 52% of their drug content after 72 h of incubation in a GSH reduction environment. The HeLa cells treated with DOX loaded MoS₂–GSH–CYS–PF127 showed 38% toxicity at drug concentration of 40 μg, which indicated that the successfully released of drug from carrier and caused the cell death. Further, fluorescence microscopy images of HeLa cells revealed the potential behavior of the MoS₂–GSH–CYS–PF12 nanocomposite during the 2- and 4 h incubation periods; the nanocomposite was only found in the cytoplasm of HeLa cells. Interestingly, after 6 h of incubation, the drug was slowly released from the nanocomposite and could enter the nucleus as confirmed by fluorescence imaging of HeLa cells. Altogether, our synthesized PF127-coated MoS₂ nanocomposite could be effectively adopted in the near future as a GSH-sensitive drug carrier.

In this study, bio-responsive polymeric MoS₂ nanocomposites were prepared for use as a drug carrier for cancer therapy.     

Doxorobicin as cargo in a redox-responsive drug delivery system capped with water dispersible ZnS nanoparticles

In this work, we have prepared and investigated a redox-responsive drug delivery system (DDS) based on a porous carrier. Doxorubicin (DOX), a chemotherapy medication for treatment of different kinds of cancer, was used as a model drug in the study. DOX was loaded in ordered hexagonal mesoporous silica SBA-15, a nanoporous material with good biocompatibility, stability, large pore size and specific surface area (S_BET = 908 m² g⁻¹, V_P = 0.79 cm³ g⁻¹, d = 5.9 nm) and easy surface modification. To prepare the redox-responsive system, cystamine derivative ligands, with redox active disulphide linkers were grafted onto the surface of SBA-15. To ensure no significant premature release of DOX from the porous system, thioglycolic acid modified ZnS nanoparticles (ZnS–COOH NPs) were used as pore capping agents. The grafted redox-responsive cystamine derivative ligand containing disulphide linkers was bonded by a peptide bond to the thioglycolic acid groups of ZnS–COOH NPs, capping the pores. Once the disulphide bond was cleaved, the ZnS–COOH NPs caps were released and pores were opened to deliver the DOX cargo. The dithiol bond was cleavable by redox active molecules such as dithiothreitol (DTT) or glutathione, the concentration of which in cancer cells is 4 times higher than in healthy cells. The redox release of DOX was studied in two different media, physiological saline solution with DTT and saline without DTT. The prepared DDS proved the concept of redox responsive release. All samples were characterised by powder X-ray diffraction (XRD), transition electron microscopy (TEM), nitrogen adsorption/desorption at 77 K, Fourier-transform infrared spectroscopy (FTIR), thermal analysis and zeta potential measurements. The presence of semiconducting ZnS nanoparticle caps on the pore openings was detected by magnetic measurements using SQUID magnetometry showing that such cargo systems could be monitored using magnetic measurements which opens up the possibilities of using such drug delivery systems as theranostic agents.

Redox-responsive drug delivery system was studied. ZnS nanoparticles served as pore capping agent to prevent premature release of anticancer drug. Such cargo can be monitored by magnetic field which opens possibilities its use in theranostics.     

Indirect fabrication of versatile 3D microfluidic device by a rotating plate combined 3D printing system

In the past decade, 3D-printing technology has been applied in the field of microfluidics to fabricate microfluidic devices for wide-ranging areas of study including chemistry, biology, medicine, and others. However, these methods face several limitations such as insufficient resolution and long fabrication time. In this study, versatile microfluidic devices with different functions were indirectly fabricated by a rapid sacrificial template printing process using a photocurable fluoropolymer with chemical durability. The Pluronic® F127 hydrogel as a sacrificial template was rapidly patterned on substrates by a non-lithographic printing process using a computer-controlled 3D-printing system. Viscous fluoropolymer was cast on the non-deformable template that was consequently removed by applying heat and negative pressure after UV curing. The chemical-resistant and transparent microchannels were oblate-hemispherical on the cross section. They were tested by performing a heterogeneous catalytic reaction as well as a photochemical reaction. The microchannels with controlled heights were devised to induce convection for functioning as a micromixer with asymmetric flows. Moreover, upon printing the Pluronic® F127 on both sides of the PFPE (perfluoropolyether–urethane dimethacrylate) membrane substrate, the 3D hybrid microfluidic device was embedded with a permeable membrane between the lower and upper channels, which is useful for gas–liquid chemical processes.

Rapid on-demand sacrificial printing techniques using suitable combinations of resin and sacrificial materials would be desirable to fabricate versatile and functional microfluidic devices with complex designs and chemical resistance.     

pH-Responsive hyperbranched polypeptides based on Schiff bases as drug carriers for reducing toxicity of chemotherapy

Polymeric micelles have great potential in drug delivery systems because of their multifunctional adjustability, excellent stability, and biocompatibility. To further increase the drug loading efficiency and controlled release ability, a pH-responsive hyperbranched copolymer methoxy poly(ethylene glycol)-b-polyethyleneimine-poly(Nε-Cbz-l-lysine) (MPEG-PEI-PBLL) was synthesized successfully. MPEG-PEI-NH₂ was synthesized to initiate the ring-opening polymerization of benzyloxycarbonyl substituted lysine N-carboxyanhydride (Z-lys NCA). The introduction of Schiff bases in the polymer make it possible to respond to the variation of pH values, which cleaved at pH 5.0 while stable at pH 7.4. As the polymer was amphiphilic, MPEG-PEI-PBLL could self-assemble into micelles. Owing to the introduction of PEI, which make the copolymer hyperbranched, the pH-responsive micelles could efficiently encapsulate theranostic agents, such as doxorubicin (DOX) for chemotherapy and NIRF dye DiD for in vivo near-infrared (NIR) imaging. The drug delivery system prolonged the drug circulation time in blood and allowed the drug accumulate effectively at the tumor site. Following the guidance, the DOX was applied in chemotherapy to achieve cancer therapeutic efficiency. All the results demonstrate that the polymer micelles have great potential for cancer theranostics.

Polymeric micelles have great potential in drug delivery systems because of their multifunctional adjustability, excellent stability, and biocompatibility.     

Upregulation of CD3ζ and L‐selectin in antigen‐specific cytotoxic T lymphocytes by eliminating myeloid‐derived suppressor cells with doxorubicin to improve killing efficacy of neuroblastoma cells in vitro

BackgroundAgglomeration of myeloid‐derived suppressor cells (MDSCs) in tumors impedes immunotherapeutic effects. Doxorubicin (DOX) is currently the most specific drug used for the selective removal of MDSCs. Here, we study the feasibility and mechanism of eliminating MDSCs by DOX to improve antigen‐specific cytotoxic T lymphocyte (CTL)‐killing neuroblastoma (NB) cells in vitro.MethodsCTL and MDSC were prepared; then, CTLs, NB cells, MDSCs, and DOX were mixed and cultivated in different collocation patterns and divided into different groups. The levels of cluster of differentiation 3ζ chain (CD3ζ) and L‐selectin in CTL in different groups were detected. Thereafter, the killing rate of NB cells and secretion of interleukin‐2 and interferon‐γ were measured and compared.ResultsBy real‐time polymerase chain reaction (PCR) and Western blot test respectively, the proliferation and killing effect of CTLs on NB cells were all inhibited by MDSC through downregulating CD3ζ (p = 0.002; p = 0.001) and L‐selectin (p = 0.006; p < 0.001). However, this inhibitory effect was reversed by DOX. Significant differences were observed in the levels of interleukin‐2 (p < 0.001), interferon‐γ (p < 0.001), and the killing rate (p < 0.001) among the groups, except between the CTL +SK‐N‐SH and CTL +SK‐N‐SH +DOX groups (p > 0.05).ConclusionsTargeted elimination of MDSCs by DOX can improve Ag‐specific CTL killing of NB cells in vitro by upregulating CD3ζ and L‐selectin. This study provides a novel method to enhance the immunotherapeutic effects of NB.     

Doxorubicin-loaded mesoporous magnetic nanoparticles to induce apoptosis in breast cancer cells

Selective targeting of chemotherapeutic drugs toward the cancer cells overcomes the limitations involved in chemotherapy. Ideally, targeted delivery system holds great potential in cancer therapy due to specific release of drug in the cancer tissues. In this regard, DOX-loaded chitosan coated mesoporous magnetic nanoparticles (DOX-CMMN) were prepared and evaluated for its physicochemical and biological characteristics. Nanosized magnetic nanoparticles were observed with a high loading capacity for DOX. The drug-loaded nanoparticles exhibited a controlled and sustained release of drug without any burst release phenomenon. The DOX-DMMN showed a concentration-dependent cell proliferation inhibitory action against breast cancer cells. The blank nanoparticles showed excellent biocompatibility with cell viability >85% at the maximum tested concentration. Our results showed that chitosan coated magnetic system has high potential for breast cancer targeting under an alternating current magnetic field (ACMF). The present study showed that magnetic nanoparticles can be targeted to tumor cells under the presence of oscillating magnetic field. The combined effect of chemotherapy and thermotherapy can have a promising clinical potential for the treatment of breast cancer.     

Development of a pH-sensitive functionalized metal organic framework: in vitro study for simultaneous delivery of doxorubicin and cyclophosphamide in breast cancer

Exploration of an efficient dual-drug based nanocarrier with high drug loading capacity, specific targeting properties, and long-term stability is highly desirable in cancer therapy. Metal–organic frameworks (MOFs) have proven to be a promising class of drug carriers due to their high porosity, crystalline properties with defined structure information, and their potential for further functionalization. To enhance the drug efficacy as well as to overcome the burst effect of drugs, here we synthesized a pH responsive folic acid (FA) and graphene oxide (GO) decorated zeolitical imidazolate frameworks-8 (GO–FA/ZIF-8), for targeted delivery of doxorubicin (DOX) and cyclophosphamide (CP), simultaneously. In this system, DOX molecules were encapsulated in the pores of ZIF-8 during in situ synthesis of ZIF-8 and CP molecules have been captured by the GO surface via hydrogen bonding and π–π interactions as well. Furthermore, the resulting pH-responsive nanocarrier (DOX@ZIF-8/GO–FA/CP) showed in vitro sustained release characteristics (76% of DOX and 80% of CP) by cleavage of chemical bonding and disruption of the MOFs structure under acidic condition (at pH 5.6). Moreover, DOX@ZIF-8/GO–FA/CP has synergistic cytotoxic effects as compared to the combination of both the drugs without ZIF-8/GO–FA when treating MCF-7 and MDA-MB-231 breast cancer cell lines (with a combination index of 0.29 and 0.75 for MCF-7 and MDA-MB-231 cell-lines, respectively). Hence this system can be applied as an effective platform for smart dual drug delivery in breast cancer treatment through its remarkable manageable multidrug release.

Exploration of an efficient dual-drug based nanocarrier with high drug loading capacity, specific targeting properties, and long-term stability is highly desirable in cancer therapy.     

UCN–SiO2–GO: a core shell and conjugate system for controlling delivery of doxorubicin by 980 nm NIR pulse

Herein, graphene oxide (GO) has been attached with core–shell upconversion-silica (UCN–SiO₂) nanoparticles (NPs) to form a GO–UCN–SiO₂ hybrid nanocomposite and used for controlled drug delivery. The formation of the nanocomposite has been confirmed by various characterization techniques. To date, a number of reports are available on GO and its drug delivery applications, however, the synergic properties that arise due to the combination of GO, UCNPs and SiO₂ can be used for controlled drug delivery. New composite UCN@SiO₂–GO has been synthesized through a bio-conjugation approach and used for drug delivery applications to counter the lack of quantum efficiency of the upconversion process and control sustained release. A model anticancer drug (doxorubicin, DOX) has been loaded to UCNPs, UCN@SiO₂ NPs and the UCN@SiO₂–GO nanocomposite. The photosensitive release of DOX from the UCN@SiO₂–GO nanocomposite has been studied with 980 nm NIR laser excitation and the results obtained for UCNPs and UCN@SiO₂ NPs compared. It is revealed that the increase in the NIR laser irradiation time from 1 s to 30 s leads to an increase in the amount of DOX release in a controlled manner. In vitro studies using model cancer cell lines have been performed to check the effectiveness of our materials for controlled drug delivery and therapeutic applications. Obtained results showed that the designed UCN@SiO₂–GO nanocomposite can be used for controlled delivery based therapeutic applications and for cancer treatment.

A GO–UCN–SiO₂ hybrid nanocomposite for loading of doxorubicin and its use in in vitro efficiency for killing carcinoma cells.     

Co-delivery of dihydroartemisinin and docetaxel in pH-sensitive nanoparticles for treating metastatic breast cancer via the NF-κB/MMP-2 signal pathway

Metastasis is a major barrier in cancer chemotherapy. Prolonged circulation and rapid, specific intracellular drug release are two main goals in the development of nanoscale drug delivery systems to treat metastatic breast cancer. In this study, we investigated the anti-metastasis effect of docetaxel (DTX) in combination with dihydroartemisinin (DHA) in metastatic breast cancer 4T1 cells. We synthesized a pH-sensitive material 4-arm-PEG-DTX with a hydrazone bond and used it to construct nanoparticles that co-deliver DTX and DHA (D/D NPs). The D/D NPs had a mean size of 142.5 nm and approximately neutral zeta potential. The pH-sensitive nanoparticles allowed acid-triggered drug release at the tumor site, showing excellent cytotoxicity (IC50 = 7.0 μg mL⁻¹), cell cycle arrest and suppression of cell migration and invasion. The mechanisms underlying the anti-metastasis effect of the D/D NPs involved downregulation of the expression of p-AKT, NF-κB and MMP-2. Therefore, D/D NPs represent a new nanoscale drug delivery system for treating metastatic breast cancer, responding to the acidic tumor microenvironment to release the chemotherapeutic drugs.

Co-delivery DTX and DHA as acid-sensitive nanoparticles to exert synergistic effects for metastatic breast cancer therapy.     

Bifunctional folic-conjugated aspartic-modified Fe3O4 nanocarriers for efficient targeted anticancer drug delivery

Functionalization of nanocarriers has been considered the most promising way of ensuring an accurate and targeted drug delivery system. This study reports the synthesis of bifunctional folic-conjugated aspartic-modified Fe₃O₄ nanocarriers with an excellent ability to deliver doxorubicin (DOX), an anticancer drug, into the intercellular matrix. Here, the presence of amine and carboxylate groups enables aspartic acid (AA) to be used as an efficient anchoring molecule for the conjugation of folic acid (FA) (EDC–NHS coupling) and DOX (electrostatic interaction). Based on the results, surface functionalization showed little effect on the physicochemical properties of the nanoparticles but significantly influenced both the loading and release efficiency of DOX. This is primarily caused by the steric hindrance effect due to large and bulky FA molecules. Furthermore, in vitro MTT assay of B16–F1 cell lines revealed that FA conjugation was responsible for a significant increase in the cytotoxicity of DOX-loaded nanocarriers, which was also found to be proportional to AA concentration. This high cytotoxicity resulted from an efficient cellular uptake induced by the over-expressed folate receptors and fast pH triggered DOX release inside the target cell. Here, the lowest IC₅₀ value of DOX-loaded nanocarriers was achieved at 2.814 ± 0.449 μg mL⁻¹. Besides, further investigation also showed that the drug-loaded nanocarriers exhibited less or no toxicity against normal cells.

Aspartic acid was used as an anchoring molecule for the conjugation of folic acid and doxorubicin to Fe₃O₄ nanoparticles. The as-prepared bifunctional folic-conjugated aspartic-modified Fe₃O₄ nanocarrier was shown to be as an efficient targeted anticancer drug delivery.     

Enzymatic synthesis of selenium-containing amphiphilic aliphatic polycarbonate as an oxidation-responsive drug delivery vehicle

Although functional aliphatic polycarbonates (APCs) have attracted prominent research interest as stimuli-responsive biomaterials, the majority of functional APCs are fabricated by detrimental organometallic catalysts or organo-catalysts. Herein, a facile synthetic strategy based on enzymatic polymerization was developed to construct a selenium-containing amphiphilic aliphatic polycarbonate (mPEG-b-CMP₄₅). Specifically, the selenium in its backbone framework underwent a hydrophobic–hydrophilic transition upon exposure to the abnormal ROS level of the tumor, thus providing a promising platform for ROS-triggered drug release. This amphiphilic mPEG-b-CMP₄₅ efficiently encapsulated doxorubicin (DOX) via self-assembly in aqueous solution and showed an excellent ability to regulate the release of DOX in response to H₂O₂ at biologically relevant concentrations (100 μM). These DOX-loaded nanoparticles could easily be internalized into U87 cells and possess the inherent antitumor properties of DOX, while they exhibited much lower cytotoxicity in normal cells HL-7702. Moreover, in many cases, the introduction of selenium caused high cytotoxicity of the materials, but the cytotoxicity results in HL-7702 cells demonstrated the good biocompatibility of mPEG-b-CMP₄₅. These collective data suggested the potential use of mPEG-b-CMP₄₅ as a biocompatible and smart drug delivery vehicle.

A facile synthetic strategy based on enzymatic polymerization was developed to construct a ROS-responsive polycarbonate served as biocompatible drug vehicle.     

P1c peptide decorated liposome targeting αvβ3-expressing tumor cells in vitro and in vivo

Integrin αvβ3 is a promising target for integrin-rich tumor and neovascular. In the present study, we prepared a doxorubicin (DOX)-loaded liposome of which the surface was decorated with PEG and a novel αvβ3 targeting peptide of P1c. The in vitro targeting efficiency was evaluated in αvβ3-positive (U87MG) and -negative (MCF-7) tumor cells by flow cytometry and laser confocal scanning microscopy. The in vivo therapeutic effects were evaluated in the glioblastoma U87MG-tumor bearing mouse model. The results indicated that the prepared liposomes showed mean sizes of 131.2 and 128.4 nm in diameter for P1c-modified targeting liposomes (P1c-DOXL) and non-targeting liposomes (DOXL), respectively. The DOX encapsulation efficiencies were more than 95% in both types of liposomes. The conjugation ratio for P1c decoration was 66.8%. The flow cytometry and confocal laser-scanning microscopy experiments consistently showed that the intracellular fluorescence intensity of the P1c-modified targeted liposome group was stronger than that of the non-targeted liposome group (P < 0.05) in U87MG cells. In vivo results revealed that compared with DOX or DOXL treatment, P1c-DOXL dramatically reduced tumor growth (P < 0.05) and tumor angiogenesis while much lower hepatotoxicity was observed. P1c-modified targeting liposome exhibited sustained release, enhancing the antitumor effect of DOX through targeting tumor cells and neovascular where integrin αvβ3 was overexpressed. The results indicated that P1c might be promising for active targeting delivery in cancer therapy.

A novel peptide of P1c decorated liposomes targets an integrin αvβ3 expressed tumor.     

Designing a high-performance smart drug delivery system for the synergetic co-absorption of DOX and EGCG on ZIF-8

Due to the extreme pore volume and valuable surface area, zeolitic imidazole frameworks (ZIFs) are promising vehicles that enhance the delivery of therapeutic agents to tissues. Furthermore, these nanoporous materials have high stability in the pH and temperature of the surrounding healthy cells (37 °C and pH = 7) and an exotic potential to deform in carcinogenic environment (T > 37 °C and pH ∼ 5.5), which make them perfect smart drug delivery vehicle candidates. In this work, a series of molecular dynamics (MD) and metadynamics simulations have been performed to gain molecular insight into the mechanisms involved in the process of co-loading of doxorubicin (DOX) and EpiGalloCatechin-3 Gallate (EGCG) on ZIF-8, which form a smart drug delivery system (SDDS). The obtained results revealed that DOX was adsorbed on the carrier mostly through electrostatic interactions (E_coul = ∼−1200 kJ mol⁻¹, E_tot = −1700 kJ mol⁻¹), and EGCG was stacked on ZIF-8 mainly via van der Waals interactions (E_L-J = ∼−600 kJ mol⁻¹, E_tot = ∼−1200 kJ mol⁻¹). It is worth mentioning that the drug–drug L-J interactions (E_L-J = ∼500 kJ mol⁻¹) were also important in the co-loading process. The insertion of DOX and EGCG as additive agents to the initial ZIF-8/EGCG and ZIF-8/DOX systems led to the enhancement of the drug–carrier pair interactions to about ∼−2300 kJ mol⁻¹ and ∼−2000 kJ mol⁻¹, respectively. This finding implied that the drug–drug interactions had a complementary role in the development of SDDS via ZIF-8. From the metadynamics simulation, it was found that the geometry of the drugs is a determining factor in an efficient co-loading SDDS.

Adsorption free energy of a molecule depends on where and how the molecule meets ZIF-8 surface.     

A unimolecular theranostic system with H2O2-specific response and AIE-activity for doxorubicin releasing and real-time tracking in living cells

A theranostic drug delivery system composed of tetraphenyl-ethene (AIEgen), benzyl boronic ester (trigger), and doxorubicin (drug) was designed and synthesized; its utilities for cell imaging, drug delivery tracking, and cancer cell cytociding were evaluated.

A theranostic drug delivery system composed of tetraphenylethene (AIE-gen), benzyl-boronic ester (trigger) and doxorubicin (drug) was synthesized and its functions in cell imaging, drug delivery tracking, and therapeutic effect were evaluated.

Cancer is a disease that heavily threatens human lives and chemotherapy is known to be an effective route for cancer treatment.^1,2 To achieve good therapeutic efficiency, controlled drug delivery systems are usually used in chemotherapy. Traditional drug delivery systems are nanocarriers that could specifically respond to a certain physiological environment (e.g., redox, oxidation, pH, etc.), and release the drug at the same time.^3–5 Recently, some theranostic drug delivery systems have been developed and attracted broad attention.^6–9 Theranostic drug delivery systems that integrate diagnosis and therapy often respond to cancer-associated stimuli, release the drug, and simultaneously reveal an output signal change which can be tracked in a real time manner.^6–9Fluorescent probing is one of the mostly used signaling techniques to display signal change upon drug release. Recently, fluorescent probes with AIE (aggregation-induced emission) property, namely AIEgens, have attracted great attention in both fundamental research and industrial applications.^10–16 AIEgens are non-emissive when molecularly dissolved in solution but can be induced to emit strongly in aggregate state because of the restricted intramolecular motions (RIM).^10–16 This unique RIM mechanism allows AIEgens to emit efficiently at high concentrations and in solid state. Compared with conventional dyes with aggregation caused quenching (ACQ) property, AIEgens have shown higher photo-bleaching resistance and emission stability. In addition, they have shown lower cytotoxicity than inorganic quantum dots. Thus they are ideal candidates for cell imaging and drug delivery tracking.^13–16The application of AIEgens to real-time imaging and tracking drug release process emerged as a novel strategy. For example, Liu et al. developed a light-up theranostic agent by tactfully linking a silole-based AIEgen, a cyclic RGD cancer cell-targeting peptide, a platinum Pt(iv) prodrug, and a stimulus responsive peptide together.¹⁷ The reductant of ascorbic acid in U87-MG cancer cells reduced the prodrug Pt(iv) to the active Pt(ii) drug, which triggered apoptosis by activating the pro-apoptotic enzyme caspase-3. Subsequently, the enzyme cleaved the peptide to release the AIEgens, which aggregated in the cytoplasm and emitted strongly to report the drug-induced apoptosis. Using this strategy, another light-up theranostic agent containing a tetraphenylethene (TPE) AIEgen, a RGD peptide, a Pt(iv) prodrug and a stimulus-responsive peptide was designed, and it showed evident AIE-characteristics and expected cytotoxicity for breast cancer cells over normal cells. In a later design, both prodrugs of Pt(iv) and doxorubicin (DOX) were introduced into the light-up theranostic agent, which enhanced the apoptosis of cancer cells by the synergistic effect of the two drugs.¹⁸ In 2014, Zou, Liang and colleagues reported a fluorescence based self-indicating drug delivery system, which is capable of signaling spatiotemporal drug release with TPE and DOX composite nanoparticles (NPs).⁵ The emission of TPE molecules in the composite NPs was partially quenched by the DOX aggregates via a fluorescence resonance energy transfer (FRET) mechanism. After being taken up into lysosomes, the low internal pH triggered the detachment of DOX from the composite NPs and simultaneously enhanced the emission from AIEgens due to the absence of FRET process. This drug delivery mode can track the sub-cellular location of the delivery system and the drug releasing site.In this work, we developed a theranostic drug delivery system different from those reported in the literature. The system is constructed from an AIEgen (carboxylated TPE), a benzyl-boronic ester (BBE), and a DOX prodrug. It is abbreviated as ABD-system, and its working mechanism is illustrated in Scheme 1. In comparison with the previous work, a prime difference in our design lies on the BBE trigger, which is sensitive to hydrogen peroxide that is one of the cellular reactive oxygen species (ROS). ROS play key roles in controlling the function and health of cells, and alleviated ROS level has been correlated to cancerous cells.^19–24 With their high reactivity, several drug delivery systems utilized ROS-sensitive functional groups, such as BBE and 2,4-dinitro-benzene sulfonyl, as triggers to activate prodrugs.^25–28 So far, cellular ROS has never been used as the internal agent to trigger the drug release during which a fluorescence turn-on mechanism is utilized to monitor the progress in a real-time mode. When chemically bonded together via a self-immolative BBE linker,²⁹ the TPE and DOX units are close to each other. Moreover, the emission maximum (480 nm) of TPE is largely over-lapped with the absorption maximum (480 nm) of DOX. As DOX is weakly emissive due to its ACQ property, the ABD system is faintly emissive in solution or in aggregate state because of the unique emission mechanism of the AIEgen and the efficient FRET from TPE to DOX. If the ABD systems are internalized into a living cell with elevated cellular ROS level, the BBE linker would be cleaved, thereby the DOX and TPE units would be disassociated with each other and the FRET process is disrupted. As a result, DOX would be released and taken up into cell nucleus with red fluorescence. Meanwhile, the TPE molecules would form aggregate in the cellular plasma for their hydrophobic nature thereby the blue fluorescence could be detected. By monitoring the fluorescence changes in blue and red channels, the spatial and temporal information of the DOX release could be extracted.Open in a separate windowScheme 1Chemical structure of the theranostic drug delivery ABD-system and a schematic illustration of its working mechanism in a cell with elevated hydrogen peroxide level.The chemical structure of the theranostic ABD-system is shown in Scheme 1, and its synthetic route, preparation procedure, and characterization data of related intermediates are described in the Experiment section and ESI^† (Fig. S1 to S12^†). The spectral characterization results indicated that the expected ABD-system has been successfully obtained. With the ABD-system in hand, we first investigated its fluorescence responses towards hydrogen peroxide H₂O₂, in order to examine the proposed working mechanism shown in Scheme 1. The ABD-system itself in the solid state was weakly red emissive under the excitation of UV light due to efficient FRET, in spite of the strong emission of the TPE unit in solid state. In PBS buffer, almost no emission was recorded for the ABD-system upon excitation at 330 nm (Fig. 1), because the fluorescence of TPE is absorbed by DOX, while DOX is weakly fluorescent in aggregate state (the size distribution of the aggregates is shown in Fig. S13^†). After the addition of H₂O₂ (100 μM) into buffer solution, blue fluorescence was observed and the brightness enhanced gradually with time. As shown in Fig. 1A and B, the emission peak appeared at around 480 nm, which was assigned to the emission from the aggregates of hydrophobic carboxylated TPE molecules, as indicated by its AIE property (Fig. S15 and S16^†). This is because, when the BBE unit is oxidized by H₂O₂, the linkage between TPE and DOX is broken, and DOX and carboxylated TPE are released (cf. Fig. S17^†). The hydrophobic carboxylated TPE molecules form aggregates which emit blue fluorescence, while the DOX molecules are soluble in the buffer solution and emit weak red fluorescence. Since the distance between carboxylated TPE and DOX is far enough, FRET process is prohibited and hence the blue emission has been observed.Open in a separate windowFig. 1(A) Changes in fluorescence (FL) spectra of the ABD-system (10 μM) in PBS buffer solution in the presence of H₂O₂ (100 μM) for different times (0 to 90 min). (B) Plot of FL intensity versus incubation time. The data are extracted from the spectra in (A). (C) FL spectra of the ABD-system in PBS buffer solution incubated with different concentrations of H₂O₂ for 90 min; (D) plot of the relative FL intensity versus H₂O₂ concentration. The data are extracted from (C), and the inset photographs show the FL images of the stock solutions with and without H₂O₂. Concentration of the ABD-system (10 μM); PBS buffer solution (pH = 7.8, 10 mM, containing 1% DMSO); temperature: 37 °C; excitation wavelength: 330 nm for FL measurement and 365 nm for photograph.As indicated by the experimental data shown in Fig. 1A and B, the emission intensity increased gradually and the spectral features reached a steady state (unchanged) after 80 min. The experimental results shown in Fig. 1C and D were obtained by reacting with H₂O₂ for 90 min. A prominent dose-dependent behavior is observed. The emission intensity continuously increases with the increasing H₂O₂ concentration from 10 to 90 μM, and the photograph clearly displays the blue emission from the AIEgen (inset of Fig. 1D). A fluorescence intensity enhancement of 142-fold was observed when the H₂O₂ concentration was increased to 90 μM.Considering that other ROS, RNS (reactive nitrogen species) and RSS (reactive sulphur species), such as O₂⁻, GSH, ClO⁻, ·OH, ¹O₂, tert-butyl hydroperoxide (TBHP), alkylperoxyl radical (ROO·), NO₂⁻, NO₃⁻, and S₂⁻, may coexist with H₂O₂ in physiological environment, we also examined the fluorescent response of the ABD-system to the interfering species. As shown in Fig. 2, except for H₂O₂, all of the other reactive species can only lead to very small changes in fluorescence intensity under the identical experimental condition. Such an excellent selectivity is ascribed to the rational molecular design, since among the ROS we tested, only H₂O₂ can react with the BBE unit to induce the separation between TPE and DOX units and then turn on fluorescence emission.Open in a separate windowFig. 2FL response of the ABD system (10 μM) to a series of ROS (100 μM) in PBS buffer solution. I₀ and I are the FL intensity of the ABD-system in PBS buffer solution in the presence of ROS, RNS, and RSS including H₂O₂, O₂⁻, GSH, ClO⁻, ·OH, ¹O₂, TBHP, ROO·, S₂⁻, NO₂⁻ and NO₃⁻. PBS buffer solution (pH = 7.8, 10 mM, containing 1% DMSO); temperature: 37 °C; excitation wavelength: 330 nm; reaction time: 100 min.Based on the above results, the ABD system works well in buffer solutions. The blue emission from the aggregate of carboxylated TPE can be specifically triggered by H₂O₂, indicating the releasing of DOX. To check whether this system can work well in living cells, we incubated HeLa cells in different conditions and monitored the fluorescence from the cells using confocal laser scanning microscopy (CLSM) technique. When HeLa cells were loaded with 10 μM ABD-system and incubated at 37 °C for 2 h, no fluorescence emission could be recorded in blue channel (420–540 nm), but red emission with moderate intensity was recorded in the spectral window of 550–650 nm. The overlay (Fig. 3D) of the bright field (Fig. 3C) with the fluorescent images suggest that the ABD-system is uniformly distributed in the cytoplasm.Open in a separate windowFig. 3Confocal images of HeLa cells after incubation with TPE–DOX with different treatment; (A–D) no more treatment; (E–H) cells treated with PMA for 30 min and incubated for another 2.5 hour; (I–L) cell treated with PMA for 30 min and incubated for another 5.5 hour. Blue channel: excitation wavelength: 405 nm; collection wavelength: 420–540; red channel: excitation wavelength: 488 nm; collection wavelength: 550–650 nm. All images share the same scale bar (20 mm).When ABD-system loaded HeLa cells were treated with 5 μg mL⁻¹ phorbol-12-myristate-13-acetate (PMA) for 30 min and then incubated for another 2.5 hours, evident blue emission was detected in spectral window of 420–540 nm (Fig. 3E). Meanwhile, red emission was also collected in the wavelength range of 550–650 nm (Fig. 3F). Since PMA is an agent widely used to in situ induce the generation of H₂O₂ in living cells, the introduction of PMA can effectively elevate the H₂O₂ level and thereby trigger the release of both carboxylated TPE and DOX in the cytoplasm. The generated carboxylated TPE molecules were insoluble in aqueous medium (e.g., cytoplasm), and hence formed aggregates. Due to the inhibited FRET process induced by DOX leaving and the TPE aggregate formation, strong blue fluorescence from TPE donor was recorded according to the RIM mechanism. At the same time, DOX molecules were also released into cytoplasm. The merged image indicates that the blue and red emissions from the aggregates of the carboxylated TPEs and DOXs have good overlap and are distributed all over the cytoplasm.After further incubation for additional 3 h, the blue emission still existed in the cytoplasm (Fig. 3I), while the red emission could be observed in the cell nucleus (Fig. 3J). This indicates that some DOX molecules are translocated into cell nucleus. This observation is reasonable because DOX is a drug that works on DNA. In the overlay image (Fig. 3L), the entities with purple fluorescence are assigned to cell nucleus, indicative of the colour mixing of blue and red emissions. The surrounding blue fluorescence comes from the aggregated carboxylated TPEs.In order to evaluate therapeutic performance of the ABD system, cell viability was studied using MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. As shown in Fig. 4, without the ABD system, the cell viability is as high as 98% in the presence of PMA under standard incubation condition. PMA itself has no therapeutic effect on HeLa cells. Without the treatment of PMA, the presence of ABD system could not induce the therapeutic effect, and the cell viabilities in different concentrations and incubation time were almost the same, as suggested by the data in Fig. 4. In sharp contrast, when activated with PMA, cell viabilities decreased dramatically and synergistically with the increment of prodrug concentration and incubation time. As a direct evidence, the bright field images (Fig. 3C, G and K) and the confocal fluorescent images demonstrate that the morphologies of HeLa cells changed significantly upon treated with ABD and PMA for hours. These results strongly prove that ABD system has good therapeutic effects on HeLa cells.Open in a separate windowFig. 4Dose-dependent cytotoxicity of ABD system to HeLa cells with and without PMA treatments, which were estimated by using standard MTT assay.In summary, an ABD-system consisting of carboxylated TPE (AIEgen), active linker (benzyl-boronic ester) and DOX (drug) moieties has been designed, synthesized and characterized. Its drug releasing and fluorescent tracking processes and its therapeutic effect have been studied. In living HeLa cells incubated with the ABD-system, sole red emission can be observed. After treatment with PMA, H₂O₂ is generated in situ and it reacts with the benzyl-boronic ester moiety, leading to the decomposition of the ABD system and the spatial separation of carboxylated TPE and DOX moieties. As a result, both of the blue emission from the AIEgen and red emission from DOX molecules have been monitored in the respective spectral channels, due to restricted FRET between AIEgens and DOX. Based on the dual emission colors, the DOX releasing can be monitored. After a short period of time, the released DOX molecules enter into nucleus to realize its therapeutic function, which is revealed by the observation of the red emission in the nucleus region. The therapeutic effect has been estimated by the MTT experiments. In addition, the ABD system shows high stability in the pH range from 4 to 9 and good selectivity over other interfering reactive species including OCl⁻, O₂⁻, ·OH, GSH, ¹O₂, TBHP, ROO·, NO₂⁻, NO₃⁻ and S₂⁻, which are also involved in biological systems.