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We report the synthesis and self-assembly of a library of squaramide-based bolaamphiphiles with variable hydrophobic and hydrophilic domain sizes, consisting of varied aliphatic chains (n = 2 to 12 methylene repeat units) and linear oligo(ethylene glycol) (m = 11 to 36 repeat units), to understand their effect on the formation of supramolecular polymer nanoparticles. Systematic variation of the hydrophobic chain length show that a minimum hydrophobic domain is required to shield the squaramide units from water when a constant hydrophilic domain is maintained for self-assembly. By contrast, significant increases to the hydrophilic chain length of the bolaamphiphile, while keeping the hydrophobic domain constant, results in a transition from fibrillar to spherical nanoscale objects with an alteration in the aggregation mode of the monomers likely due to steric constraints of the oligo(ethylene glycol) chains. By understanding the self-assembly space achievable for these squaramide-based bolaamphiphiles through examining the interplay between various monomer features, we show their distinct effects on the formation of self-assembled nanoparticles with possibilities to modulate their shape and size in water for future applications in the biomedical area.

Previously developed Asn-Gly-Arg (NGR) peptide-modified multifunctional poly(ethyleneimine)–poly(ethylene glycol) (PEI–PEG)-based nanoparticles (TPIC) have been considered to be promising carriers for the co-delivery of DNA and doxorubicin (DOX). As a continued effort, the aim of the present study was to further evaluate the interaction between TPIC and human umbilical vein endothelial cells (HUVEC) to better understand the cellular entry mechanism. In the present investigation, experiments relevant to co-localization, endocytosis inhibitors and factors influencing the internalization were performed. Without any treatment, there was no co-localization between aminopeptidase N/CD13 (APN/CD13) and caveolin 1 (CAV1). However, co-localization between CD13 and CAV1 was observed when cells were incubated with an anti-CD13 antibody or TPIC. As compared with antibody treatment, TPIC accelerated the speed and enhanced the degree of co-localization. TPIC entered HUVEC not only together with CD13 but also together with CAV1. However, this internalization was not dependent on the enzyme activity of CD13 but could be inhibited by methyl-β-eyclodextfin (MβCD), further identifying the involvement of caveolae-mediated endocytosis (CvME). This conclusion was also verified by endocytosis inhibitor experiments., Graphical abstract Asn-Gly-Arg (NGR)-functioned multifunctional poly(ethyeneimine)–poly(ethyleneglycol) (PEI–PEG)-based nanoparticles (TPIC) could influenced the distribution of CD13 on human umbilical vein endothelial cells through the interaction between CD13 and NGR, causing CD13 clustering, leading to the co-localization of CD13 and caveolin 1, and finally induce the internalization via the caveolea-mediated endocytosis.fx1

Chunxi Liu,1,* Tingxian Liu,2,* Yongjun Liu,2 Na Zhang2 1Department of Pharmacy, Qilu Hospital, Shandong University, Ji’nan, China; 2School of Pharmaceutical Science, Shandong University, Ji’nan, China *These authors contributed equally to this work Background: We previously developed a simple effective system based on oligodeoxynucleotides with CGA repeating units (CGA-ODNs) for Dox and siRNA intracellular co-delivery. Methods: In the present study, the in vitro cytotoxicity, gene transfection and in vivo safety of the co-delivery system were further characterized and discussed. Results: Compared with poly(ethyleneimine) (PEI), both CGA-ODNs and the pH-sensitive targeted coating, o-carboxymethyl-chitosan (CMCS)-poly(ethylene glycol) (PEG)-aspargine-glycine-arginine (NGR) (CMCS-PEG-NGR, CPN) showed no obvious cytotoxicity in 72 h. The excellent transfection capability of CPN coated Dox and siRNA co-loaded nanoparticles (CPN-PDR) was confirmed by real-time PCR and Western blot analysis.It was calculated that there was no significant difference in silencing efficiency among Lipo/siRNA, CPN-modified siRNA-loaded nanoparticles (CPN-PR) and CPN-PDR. Furthermore, CPN-PDR was observed to be significantly much more toxic than Dox- and CPN-modified Dox-loaded nanoparticles (CPN-PD), implying their higher antitumor potential. Both hemolysis tests and histological assessment implied that CPN-PDR was safe for intravenous injection with nontoxicity and good biocompatibility in vitro and in vivo. Conclusion: The results indicated that CPN-PDR could be a potentially promising co-delivery carrier for enhanced antitumor therapy. Keywords: co-delivery, doxorubicin, VEGF, cytotoxicity, transfection

The greatest challenge in combining chemotherapy and gene therapy is the construction of a suitable platform for the co-delivery of the drug and the therapeutic gene. In this study, a simplified and effective system for the co-loading and intracellular co-delivery of doxorubicin (Dox) and siRNA was developed. Oligodeoxynucleotides with CGA repeating units (CGA-ODNs) were introduced to load Dox. The loading mechanism was based on the ability of Dox to intercalate within double-stranded 5'-GC-3' or 5'-CG-3' sequences. Poly(ethyleneimine) (PEI) was used to condense siRNA and Dox loaded CGA-ODNs (CGA-ODNs-Dox) to obtain Dox and siRNA co-loaded nanocomplexes (PEI/CGA-ODNs-Dox&siRNA, PDR). The cellular uptake of PDR in A549 and HepG2 cells was 39.52% and 36.78%, respectively, indicating that the co-loading and co-delivery effect was achieved through the mono-loading method. An in vitro drug release study indicated that CMCS-poly(ethylene glycol) (PEG)-NGR (CPN) modified PDR (CPN-PDR) displayed a pH-triggered drug release property due to the reversed surface charge of CMCS in an acidic environment. Cellular uptake studies also confirmed that the disassembly of CPN-PDR was induced by an acidic pH in the extracellular matrix. Moreover, lysosomal escape of both Dox and siRNA was observed. Successful accumulation of Dox in the cell nucleus and siRNA in the cytoplasm was also demonstrated. Consequently, the novel construction of a simplified loading method and high co-delivery efficiency was proven to be a promising platform for the co-delivery of drug and siRNA.

A novel dual pH-sensitive co-delivery system, programmed to respond to tumor extracellular pH (6.0–7.0) and intracellular pH (4.5–6.5) environments, was designed for the controlled co-delivery of DOX and genes. Tumor intracellular pH-sensitive cationic doxorubicin (DOX)–poly(ethyleneimine) (PEI) conjugates (DOX–PEI, DP) were synthesized via pH-sensitive hydrazone bonds. As O-carboxymethyl-chitosan (CMCS) possesses cationic charges under pH 6.5 and is anionically charged above pH 7.0, tumor extracellular pH (6.0–7.0) triggered charge reversal CMCS–poly(ethylene glycol) (PEG)–aspargine–glycine–arginine (NGR) copolymers (CMCS–PEG–NGR, CPN) were synthesized. The two materials with different properties help to construct the dual pH-sensitive co-delivery system. First, the cationic DP interacts with anionic pDNA to form a DOX and gene co-loaded DP/pDNA (DPD) core. Then, anionic CPN is adsorbed on the surfaces of the positive charged DPD to form dual pH-sensitive CPN/DPD (CDPD). CDPD and DPD exhibited spherical shapes, uniform particle size distributions (137.4 ± 2.7 nm and 80.0 ± 4.2 nm, respectively), and positive zeta potentials (8.25 mV and 23.59 mV, respectively). Targeted cellular uptakes of CDPD were confirmed (cellular uptakes of CDPD were 92.49 ± 2.28% and 67.82 ± 0.07% in CD13-positive A549 cells and CD13-negative HepG2 cells, respectively). The dissociation of CPN from CDPD at acidic tumor tissue was evaluated (transfection efficiencies of CDPD at pH 7.4 and pH 6.0 were 11.43 ± 0.59% and 20.20 ± 1.21%, respectively). The internalized DPD programmable release of DOX and pDNA was investigated under simulated endosomal conditions. The results suggest that dual pH-sensitive CDPD was conducive to targeted delivery and the novel functional materials ensured the successful construction of novel dual pH-sensitive CDPD using a simple method.

A multifunctional pH-sensitive polymeric nanoparticle system was developed for simultaneous tumor magnetic resonance imaging (MRI) and therapy. The nanoparticles were self-assembled using the multi-block polymer poly(lactic acid)-poly(ethylene glycol)-poly(l-lysine)-diethylenetriamine pentaacetic acid (PLA-PEG-PLL-DTPA) and the pH-sensitive material poly(l-histidine)-poly(ethylene glycol)-biotin (PLH-PEG-biotin). The anti-hepatocellular carcinoma (HCC) drug sorafenib was encapsulated inside the nanoparticles. Gd ions were chelated to the DTPA groups which were distributed on the nanoparticle surface. Biotinylated vascular endothelial growth factor receptor (VEGFR) antibodies were linked to the surface biotin groups of nanoparticles through the avidin linker to form the target pH-sensitive theranostic nanoparticles (TPTN). TPTN exhibited spherical or ellipsoidal shapes, uniform particle size distribution (181.4 ± 3.4 nm), positive zeta potential (14.95 ± 0.60 mV), high encapsulation efficiency (95.02 ± 1.47%) and drug loading (2.38 ± 0.04%). The pH-sensitive sorafenib release from TPTN was observed under different pH values (47.81% at pH = 7.4 and 99.32% at pH = 5.0, respectively). In cell cytotoxicity studies, TPTN showed similar antitumor effect against HepG2 cells compared to solubilized sorafenib solution after pre-incubation in acid PBS (pH = 5.0) for 1 h in vitro (P0.05). In in vivo anti-tumor studies, TPTN showed significantly higher antitumor effect in H22 tumor (VEGFR overexpressed cell line) bearing mice compared to the solubilized sorafenib solution (oral or i.v. administration) group (P0.05). In the MRI test, the T1 relaxivity value of TPTN was 17.300 mM(-1) s(-1) which was 3.6 times higher than Magnevist® (r1 = 4.8 mM(-1) s(-1)). As a positive contrast agent, TPTN exhibited higher resolution and longer imaging time (more than 90 min) in the MRI diagnosis of tumor-bearing mice compared to Magnevist® (more than 60 min). Furthermore, histological examination of TBN (blank TPTN, without sorafenib loaded) showed no visible tissue toxicity compared to normal saline. Thus, TPTN possessed dual-loading drugs and imaging agents, active targeting and pH-triggered drug release properties in one platform with good biocompatibility. All of these results indicated that TPTN was a promising theranostic carrier which could be a platform for the development of novel multifunctional theranostic agents.