Cellular Uptake Ability Research Articles

Assemblies of Peptide‐Cytotoxin Conjugates for Tumor‐Homing Chemotherapy

AbstractPeptide‐drug conjugates are prodrugs that have the advantages of precise molecular structure and the direct exploitation of tumor‐homing, penetration or the cellular uptake abilities of the peptides such as the neuropilin‐1 receptor targeting peptide. The prodrugs generally have fast blood clearance due to their low molecular weights and thus are made to self‐assemble into nanostructures, preferably nanosized micelles and vesicles for intravenous administration, to slow their renal clearance. However, most peptidyl prodrugs usually form precipitates, irregular nanofibers or gels that are unsuitable for intravenous injection. Herein, a arginine‐glycine‐aspartic acid‐lysine (RGDK) peptide and cytotoxin 7‐ethyl‐10‐hydroxycamptothecin (SN38) are used to synthesize the tumor‐homing prodrugs (SN38‐Peps) and explore their structure–micelle formation relationships. A small library of SN38‐Peps is obtained using different structures of peptides, linkers, and drug conjugation sites, and the factors affecting the assembly of SN38‐Peps as well as the stability of formed micelles are investigated. An optimized SN38‐Pep, (MOM)SN38(20)‐CRGDK, is finally obtained which forms stable micelles with a hydrodynamic diameter around 110 nm and a fixed drug loading content as high as 35%. The micelles show a prolonged blood circulation, significantly enhanced tumor accumulation, and therefore improved anticancer activity as compared to the non‐targeting prodrug and a clinically used anticancer drug.

Upconversion Nanoparticle-Based Strategy for Crossing the Blood-Brain Barrier to Treat the Central Nervous System Disease.

The blood-brain barrier (BBB) is a major challenge for the treatment of central nervous system (CNS) diseases. The BBB strictly regulates the movement of molecules into and out of the brain, and therefore protects the brain from noxious agents. However, for this reason the BBB also acts as a major obstacle that prevents most therapeutic molecules from getting into the target site of the brain. Therefore, it is essential to develop an efficient and general approach to overcome the BBB and transport the drug to the targeted region. Nanoparticle-based drug delivery systems are emerging as a promising drug delivery platform, due to their distinct advantages of tunable biophysical properties such as surface chemistry, size, and shape leading to various biological actions (like clearance, biodistribution, and biocompatibility) in the body. Therefore, it was hypothesized that the surface and shape of nanoparticles will influence their BBB permeation efficiency. Here, we describe a series of upconversion nanoparticles with different surfaces (oleic acid-free, DNA-modified, Silica coating, and PEG-encapsulated), PEGylated UCNPs with various shapes were generated (including sphere and rod). The cellular uptake ability, biodistribution, and BBB penetration of those UCNPs were assessed in cultured cells (NSC-34 neuron- like cells) and in vivo (zebrafish models).

Highly sensitive glutathione assay and intracellular imaging with functionalized semiconductor quantum dots.

Glutathione (GSH) plays a vital role in biological systems and is associated with human pathology. The engineering of semiconductor quantum dots (QDs) as fluorescent probes for GSH sensing and bioimaging is a potential yet rarely reported approach. Herein, we report the in situ growth of manganese dioxide nanosheets (MnO2) on silica-coated semiconductor quantum dots (QD@SiO2), to prepare a stable and biocompatible fluorescent nanoprobe (QD@SiO2-MnO2) for the selective and sensitive detection of GSH. The modification of QD@SiO2 with MnO2 significantly quenched the fluorescence of CdSe/ZnS QDs, yet the addition of GSH efficiently recovered the fluorescence of the nanoprobe due to the decomposition of MnO2 by GSH. This nanosensor showed a rapid response to GSH with a low detection limit, and high selectivity towards GSH over potential interferences. Furthermore, the MnO2-engineered QDs had good biocompatibility and cellular uptake ability, and were successfully applied for the real-time imaging of intracellular GSH. We envision that semiconductor QD-based probes will stimulate the study of GSH dynamics and facilitate the understanding of GSH-related pathophysiological events.

A novel nanoparticle drug delivery system based on PEGylated hemoglobin for cancer therapy

Proteins such as albumin, gelatin, casein, transferrin, and collagen are widely used as drug delivery systems. However, only albumin-based paclitaxel (PTX) formulation Abraxane® (PTX-albumin NPs prepared by nab-technology) has been successfully developed for treating metastatic breast cancer clinically due to abundant materials, simple industrial scale-up process, and well tumor-targeting ability. Hemoglobin (Hb) is another protein used for drug delivery with similar advantages. In this study, we successfully synthesized PEG-Hb nanoparticles loading with PTX based on previously well-established acid-denatured method. PEG-Hb-PTX NPs showed enhanced cellular uptake and great cellular inhibition ability in vitro. Moreover, our animal study showed that PEGylated NPs greatly accumulated in tumor tissues and exhibited excellent anticancer activity in vivo. We found that PEG-Hb-PTX NPs possess a better in vivo antitumor effect than the commercially available Taxol® formulation. We believe that PEG-Hb has great potential as an efficient drug delivery system for further clinic study.

DNA binding and in vitro antineoplastic activity of neotype water-soluble Cu(Ⅱ)-complexes based on fluorinated benzoylhydrazone porphyrin ligands

Three water-soluble Cu(Ⅱ)-based acylhydrazone porphyrin derivatives (named as CuP1, CuP2 and CuP3) were prepared and isolated successfully. Diversiform physicochemical approaches demonstrated that all of the complexes possess excellent binding affinity with ct-DNA, especially CuP3 exhibited strongest binding affinity than its analogues. Moreover, the antiproliferative properties of all compounds in vitro in human cancer cell line (A549 and H-1975 lung cancer cells, HepG2 human hepatocarcinoma, T47D breast cancer cells) and noncancerous cell line (Hs 578Bst normal breast cells) have also been evaluated by MTT assay. Besides, an extracted method was carried out to determine the cellular uptake ability of three complexes to the tested cancer cell lines. The cytotoxicity and cellular uptake tests revealed that CuP3 exhibited the best antiproliferative activity as well as cellular absorption capacity towards H-1975 cell line. In order to better understand the effects of CuP3 on H-1975 cells, the fluorescence microscopy measurements were employed, the cell membrane damage, nuclear condensation, and apoptotic bodies (brighter fluorescence) were markedly observed after incubated with CuP3 for 48 h, suggesting that CuP3 can induce cell apoptosis on H-1975 cells significantly. In addition, the flow cytometric analysis results illustrated that the complex CuP3 mainly caused an accumulation of cells in the G2 phase of the cell cycle.

Elucidating the mode of action for thiophene-based organic D-π-A sensitizers for use in photodynamic therapy.

Photodynamic therapy (PDT) is a non-invasive, selective, and cost-effective cancer therapy. The development of readily accessible templates that allow rapid structural modification for further improvement of PDT remains important. We previously reported thiophene-based organic D-π-A sensitizers consisted of an electron-donating (D) moiety, a π-conjugated bridge (π) moiety, and an electron-accepting (A) moiety as valuable templates for a photosensitizer that can be used in PDT. Our preliminary structure-activity relationship study revealed that the structure of the A moiety significantly influences its phototoxicity. In this study, we evaluated the photoabsorptive, cellular uptake, and photo-oxidizing abilities of D-π-A sensitizers that contained different A moieties. The level of phototoxicity of the D-π-A sensitizers was rationalized by considering those three abilities. In addition, we observed the ability of amphiphilic sensitizers containing either a carboxylic acid or an amide in an A moiety to form aggregates that penetrate cells mainly via endocytosis.

Synthesis, Characterization, and Biological Study of Carboxyl- and Amino-Rich g-C₃N₄ Nanosheets by Different Processing Routes.

Graphitic-phase carbon nitride (g-C₃N₄) is a graphene analogue, which has attracted much attention for its unique physicochemical properties. The bulk g-C₃N₄ is often fragmented and ionized to nanosheets, obtaining improved performance or confer novel properties. It is reported that g-C₃N₄ nanosheets using different processing methods can be modified with different functional groups, resulting in different electrical characteristics. However, the surface charge of g-C₃N₄ nanosheets is seldom mentioned as a factor influencing their biological applications. In our present study, we prepared two kinds of uniformly dispersed g-C₃N₄ nanosheets of high quality, the carboxyl- and amino-rich g-C₃N₄ nanosheets. Although both the carboxyl- and amino-rich g-C₃N₄ nanosheets displayed good biocompatibility in vitro and in vivo, however, they are at very different levels of surface charge, which are relative to different cellular uptake, penetration mechanism and lysosome escape ability of the nanosheets.

Vitamin B12-conjugated sericin micelles for targeting CD320-overexpressed gastric cancer and reversing drug resistance.

Our previous research has introduced sericin micelles to reverse drug resistance. However, these micelles could not selectively bind to gastric cancer (GC) cells. We developed vitamin B12 (VB12) conjugated sericin micelles for targeted GC therapy. We used VB12, sericin, synthetic poly(γ-benzyl-L-glutamate) (PBLG) and paclitaxel (PTX) to develop VB12-conjugated and PTX-loaded micelles (VB12-sericin-PBLG-PTX). Then we explored their physicochemical properties, cellular uptake and antitumor mechanism. VB12-sericin-PBLG-PTX micelles were proved to be of appropriate particle size, have good dispersion and are bio-safe. Following transcobalamin II (CD320)-receptor-mediated endocytosis, these swallowed micelles with GC-targeting and enhanced cellular uptake abilities, alter mitochondrial transmembrane potential/apoptosis pathway and reverse drug resistance. VB12-sericin-PBLG-PTX micelles are promising materials for GC-targeted clinical applications.

CAGW Modified Polymeric Micelles with Different Hydrophobic Cores for Efficient Gene Delivery and Capillary-like Tube Formation.

Recently, polymeric micelles with different biodegradable hydrophobic cores, such as poly(lactide-co-glycolide) (PLGA) and poly(lactide-co-3(S)-methyl-morpholine-2,5-dione) (PLMD), have been used for gene delivery. The biodegradable hydrophobic cores should play an important role in gene delivery. However, little research has focused on selectively promoting proliferation and migration of endothelial cells (ECs) as well as vascularization by altering hydrophobic cores of polymeric micelles. Herein, we prepared two kinds of CAGW peptide (selective adhesion for ECs) modified micelles with PLGA and PLMD as hydrophobic cores, respectively, and poly(ethylene glycol) (PEG) and polyethylenimine (PEI) as mixed hydrophilic shell. Their ability of condensing pEGFP-ZNF580 (pZNF580) to form gene complexes was proved by agarose gel electrophoresis assay. MTT results showed that the relative cell viability of the micelles with PLMD cores was higher than control groups and the micelles with PLGA cores. The cellular uptake ability of these CAGW modified gene complexes was higher than the complexes without CAGW target function. A similar trend was also found in transfection tests in vitro, which further demonstrated the effect of CAGW peptide and different hydrophobic cores on gene delivery. The number of migrated cells treated by the gene complexes with PLGA cores was 82 (nontarget group) and 115 (target group), whereas the complexes with PLMD cores was 88 (nontarget group) and 120 (target group). Capillary-like tube formation of CAGW peptide modified complexes with PLMD core group was much higher (about 6 times) than the PEI(10 kDa)/pZNF580 group. These results demonstrated that transfection efficiency, cell proliferation, migration, and vascularization could be promoted by altering hydrophobic cores and CAGW modification.

PH-Responsive Nanoparticles for Controllable Curcumin Delivery: The Design of Polycation Core with Different Structures

To achieve low leakage at pH 7.4, pH-controlled drug release remains a major challenge of cancer nanomedicine. Here, pH-responsive mPEG113-b-polycaprolactone (PCL)x-b-PAEMAy (PELAx) nanoparticles (NPs)-pH7.4 with different poly(2-azepane ethyl methacrylate) (PAEMA) lengths and PELA4 nanoparticles (NPs)-pHx with different pH conditions of preparing NPs based on mPEG113-b-PCLx-b-PAEMAy are prepared to investigate influences of PAEMA lengths and pH condition of preparing NPs on properties of NPs. PELAx NPs-pH7.4 and PELA4 NPs-pHx both undergo differently sharp property changes of size, charge, pH-responsive drug release, cellular uptake, and cytotoxicity, because of the protonation of PAEMA block (pKa ≈ 6.5–6.7). Among PELAx NPs-pH7.4 and PELA4 NPs-pHx, it can be found that the NPs prepared at pKa value of PAEMA exhibited exciting pH-responsive drug release, improving cellular uptake ability and obvious high cytotoxicity toward HepG-2 cells. This system not only holds great potential for drug delivery but also provides a new strategy to prepare pH-responsive NPs.

Rod-like cellulose nanocrystal/cis-aconityl-doxorubicin prodrug: A fluorescence-visible drug delivery system with enhanced cellular uptake and intracellular drug controlled release

Rod-like nanomedicines facilitate cellular uptake. This research is aimed to develop fluorescence-visible rod-like nanomedicines with enhanced cellular uptake and intracellular drug controlled release based on cis-aconityl-doxorubicin (CAD) labeled cellulose nanocrystal rods (CNR). Particularly, CAD was synthesized by the ring-opening reaction between cis-aconitic anhydride (CAA) and the amino group of Doxorubicin (DOX). Amidation reaction occurred between the 6-carboxylic groups of CAD and the amino groups of aminated CNR to give CAD labeled CNR (CAD@CNR). Compared with CNR, CAD@CNR showed similar morphology and crystal structure. The mean length of CAD@CNR was ca. 118 nm with aspect ratio ranging from 12 to 15, facilitating their endocytosis. CAD@CNR prodrug was rather stable in pH 7.4 phosphate buffer solution but tended to be hydrolyzed to release DOX under acidic condition, due to the rapid degradation of amide bonds between DOX and cis-aconitic acid via an intramolecular acid-catalyzed mechanism. CAD@CNR prodrug showed sustained drug release profiles over 40 h, and the cumulative drug release showed a tendency to increase from 36 to 80% with the pH value decreasing from 7.4 to 5.0. The half maximal inhibitory concentration (IC50) of CAD@CNR prodrug against NCI H 460 cells without NH4Cl (lysosomotropic weak bases) pretreatment was 1.75 times higher than that with 40 mM NH4Cl pretreatment, further confirmed that the DOX release from the CAD@CNR prodrug was triggered by the low pH value of lysosome (pH 5.0). Compared with DOX·HCl, CAD@CNR prodrug showed enhanced cellular uptake ability during 12 or 24 h of incubation due to the endocytosis mechanism of CAD@CNR prodrug. After incubation with cells, CAD@CNR prodrug could be observed by using fluorescence microscope due to the red fluorescence of DOX. In a word, CAD@CNR showed great potential as fluorescence-visible drug delivery system with enhanced cellular uptake and intracellular drug release due to its rod-like morphology, suitable aspect ratio, and acid-triggered drug release.

Mesenchymal stem cells ameliorate palmitic acid induced lipotoxicity of human umbilical vein endothelial cells and high‐fat induced obese rats by suppression of endoplasmic reticulum stress

BackgroundEndothelial dysfunction caused by the high levels of plasma free fatty acids is involved in the pathogenesis of metabolic syndrome (MS). Mesenchymal stem cells (MSCs) were reported able to ameliorate MS. Here we explored the effects of MSCs on palmitic acid (PA) induced endothelial lipotoxicity and disclose the mechanism in human umbilical vein endothelial cells (HUVECs).MethodsThe primary HUVECs were treated by PA (50 μM) with or without human bone marrow derived MSCs (BMSCs) for 24 h in Transwell system at a ratio of 5:1. Cell viability and functions of HUVECs were determined. Cell apoptosis, oxidative stress, inflammation and endoplasmic reticulum (ER) stress was evaluated by qPCR, western blot or flow cytometry. Then glucose and lipid metabolism was detected by glucose uptake test, oil red O stain and relative genes expression.In vivo, Male Sprague Dawley rats were fed a normal diet or high fat diet (HFD) for 16 weeks. 2×106 MSCs were transplanted to rats via tail vein injection weekly for 2 weeks. At the 19th weeks, the rats were sacrificed and abdominal and thoracic aortas were collected for further detection.Result The cell viability of HUVECs declined to 68.7% of control after PA stimulation. However, apoptosis and ROS production was not elevated by PA, and ROS scavenger MitoTempo failed to reverse PA induced cell dysfunction. Coculture with BMSCs restored the cell viability to 99.4%, and the endothelial functions (tube formation and cell migration capacity) were obviously improved by BMSCs as well. Additionally, BMSCs robustly inhibited either mRNA or protein expression of the inflammatroy factors such as IL‐1β and IL‐8. In respect of nutrients metabolism, PA induced accumulation of cellular triglyceride and PA impaired glucose uptake ability was significantly reversed in BMSCs. qPCR found that mRNA expression of multiple genes essential for glucose, lipid and cholesterol metabolism was modulated by MSCs including Fdps, Cpt1, PPARγ, PDK4, et al. Next we found BMSCs substantially attenuated PA‐triggered ER stress, evidenced by decreased genes expression of UPR genes and protein levels of CHOP, ATF6, SREBP1, and ATF4. Furthermore, ER stressor thapsigargin (Thp) specifically induced ER stress in HUVECs and interestingly, BMSCs also reduced Thp induced ER stress. In vivo, serum levels of TG, LDL‐c and TC were reduced by 54%, 43% and 31% respectively, and HDL‐c was slightly increased by MSCs injection compared with HFD group. Glucose intolerance and hyperinsulinemia was improved as well. mRNA expression of multiple genes essential for glucose, lipid and cholesterol metabolism in thoraco‐abdominal aorta was modulated by BMSCs transplantation. Western blot showed that several ER stress related protein levels (ATF4, ATF6, SREBP, CHOP) were reduced in BMSCs groups. ConclusionBMSCs were able to ameliorate PA induced lipotoxicity and metabolic disorder in both HUVECs and HFD rats. BMSCs attenuated PA‐ or Thp‐triggered ER stress, which may contribute to the protective effects of BMSCs on lipotoxicity.Support or Funding InformationThis work was supported by the Program of National Natural Science Foundation of China (31571474) and the National Key Clinical Project.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dual redox/pH-responsive hybrid polymer-lipid composites: Synthesis, preparation, characterization and application in drug delivery with enhanced therapeutic efficacy

The development of intelligent drug delivery system that can efficiently deliver drug to targeted site and release the cargo in a controlled manner is of great significance for cancer chemotherapy. In the present study, dual redox/pH-sensitive amphiphilic copolymer, cholesterol modified poly (β-amino esters)-grafted disulfide poly (ethylene glycol) methyl ether (PAE(-ss-mPEG)-g-Chol), is successfully synthesized via Michael-type step polymerization using disulfide linkage-containing PEG segment. PEGylated lipid is introduced to fabricate the doxorubicin (DOX)-loaded hybrid polymer-lipid nanoparticles (NPs) with (DOX-HDPLNPs) or without (DOX-HPLNPs) disulfides to receive stable NPs with enhanced loading capacity and improved cellular uptake ability as well as serum stability. The HDPLNPs show not only pH-sensitivity because of poly(β-amino esters) (PAE) residues in main chain but also the redox-responsivity due to the disulfides on the side chains with PEG segments. As expected, the DOX-HDPLNPs exhibited the extended blood circulation time and higher serum stability in normal physiological environment, but once deposited in the tumor microenvironment (e.g. low pH value and high GSH concentration), they underwent a dramatic size transition and charge reversal. These features not only can facilitate the NPs accumulation through the enhanced permeability and retention (EPR) effect and endocytosis by tumor cells but also realize the drug controlled release and more efficient tumor inhibition. The in vitro and in vivo results demonstrate that the DOX-HDPLNPs possess the highest therapeutic efficacy with high cellular uptake and negligible cytotoxicity compared to the controls and free drug. Therefore, the HDPLNPs have good potential as smart delivery platforms for hydrophobic anticancer drug delivery due to high uptake by tumor cells and redox/pH-triggered drug release.

Peptide Ribonucleic Acid (PRNA)–Arginine Hybrids. Effects of Arginine Residues Alternatingly Introduced to PRNA Backbone on Aggregation, Cellular Uptake, and Cytotoxicity

Intracellular delivery is crucial not only in achieving effective oligonucleotide therapeutics but also for a wide variety of therapeutic strategies. In this study to elucidate the effects of the number of incorporated arginine residues on cell membrane permeability, we designed a series of peptide ribonucleic acids (PRNAs) that alternatingly incorporated 4, 8, or 12 arginine residues (PRR1-3) in the PRNA backbone for experimental verifications. Indeed, the cellular uptake efficiency and the aggregation properties turned out to be critical functions of the number of incorporated arginine residues. Thus, the optimized PRR2 that incorporates 8 arginines showed the most efficient cellular uptake ability, much higher than that of octaarginine, without accompanying cytotoxicity or aggregation.

Self-assembly of porphyrin-grafted lipid into nanoparticles encapsulating doxorubicin for synergistic chemo-photodynamic therapy and fluorescence imaging.

The limited clinical efficacy of monotherapies in the clinic has urged the development of novel combination platforms. Taking advantage of light-triggered photodynamic treatment combined together with the controlled release of nanomedicine, it has been possible to treat cancer without eliciting any adverse effects. However, the challenges imposed by limited drug loading capacity and complex synthesis process of organic nanoparticles (NPs) have seriously impeded advances in chemo-photodynamic combination therapy. In this experiment, we utilize our previously synthesized porphyrin-grafted lipid (PGL) NPs to load highly effective chemotherapeutic drug, doxorubicin (DOX) for synergistic chemo-photodynamic therapy.Methods: A relatively simple and inexpensive rapid injection method was used to prepare porphyrin-grafted lipid (PGL) NPs. The self-assembled PGL NPs were used further to encapsulate DOX via a pH-gradient loading protocol. The self-assembled liposome-like PGL NPs having a hydrophilic core were optimized to load DOX at an encapsulation efficiency (EE) of ~99%. The resultant PGL-DOX NPs were intact, highly stable and importantly these NPs successfully escaped from the endo-lysosomal compartment after laser irradiation to release DOX in the cytosol. The therapeutic efficacy of the aforementioned formulation was validated both in vitro and in vivo.Results: PGL-DOX NPs demonstrated excellent cellular uptake, chemo-photodynamic response, and fluorescence imaging ability in different cell lines. Under laser irradiation, cells treated with a low molar concentration of PGL-DOX NPs reduced cell viability significantly. Moreover, in vivo experiments conducted in a xenograft mouse model further demonstrated the excellent tumor accumulation capability of PGL-DOX NPs driven by the enhanced permeability and retention (EPR) effect. Through fluorescence imaging, the biodistribution of PGL-DOX NPs in tumor and major organs was also easily monitored in real time in vivo. The inherent ability of porphyrin to generate ROS under laser irradiation combined with the cytotoxic effect of the anticancer drug DOX significantly suppressed tumor growth in vivo.Conclusion: In summary, the PGL-DOX NPs combined chemo-photodynamic nanoplatform may serve as a potential candidate for cancer theranostics.

A series of two-photon absorption pyridinium sulfonate inner salts targeting endoplasmic reticulum (ER), inducing cellular stress and mitochondria-mediated apoptosis in cancer cells.

In this study, a two-photon active DiphenthioER1 was screened from the four pyridinium sulfonate salt derivatives (TriphenER1-2 and DiphenthioER1-2) for imaging endoplasmic reticulum (ER) and tracking the dynamics of the ER morphology. The photophysical properties of TriphenER1-2 and DiphenthioER1-2 were systemically investigated both experimentally and theoretically, revealing that they possess large Stokes shifts, and large two-photon absorption cross-sections from 163 GM to 2023 GM in the near infrared region using a Z-scan method by avoiding spontaneous fluorescence, deep tissue penetration, and low cell damage in living cells. Among them, the DiphenthioER1 compound was found to exhibit the highest cellular uptake ability and two-photon fluorescence signals using confocal microscopy. DiphenthioER1 successfully targeted the ER and induced ER-stress, subsequently nuclear misshaping and mitochondria-mediated apoptosis have been displayed. This study thus provides great insights into designing novel two-photon fluorescent materials with dual functionality and offers tools to understand the ER-stress related mechanism for cell and chemical biologists.

Synthesis, characterization and biological evaluation of six highly cytotoxic ruthenium(ii) complexes with 4'-substituted-2,2':6',2''-terpyridine.

Herein, six ruthenium(ii) terpyridine complexes, i.e. [RuCl2(4-EtN-Phtpy)(DMSO)] (Ru1), [RuCl2(4-MeO-Phtpy)(DMSO)] (Ru2), [RuCl2(2-MeO-Phtpy)(DMSO)] (Ru3), [RuCl2(3-MeO-Phtpy)(DMSO)] (Ru4), [RuCl2(1-Bip-Phtpy)(DMSO)] (Ru5), and [RuCl2(1-Pyr-Phtpy)(DMSO)] (Ru6) with 4'-(4-diethylaminophenyl)-2,2':6',2''-terpyridine (4-EtN-Phtpy), 4'-(4-methoxyphenyl)-2,2':6',2''-terpyridine (4-MeO-Phtpy), 4'-(2-methoxyphenyl)-2,2':6',2''-terpyridine (2-MeO-Phtpy), 4'-(3-methoxyphenyl)-2,2':6',2''-terpyridine (3-MeO-Phtpy), 4'-(1-biphenylene)-2,2':6',2''-terpyridine (1-Bip-Phtpy), and 4'-(1-pyrene)-2,2':6',2''-terpyridine (1-Pyr-Phtpy), respectively, were synthesized and fully characterized. The MTT assay demonstrates that the in vitro anticancer activity of Ru1 is higher than that of Ru2-Ru6 and more selective for Hep-G2 cells than for normal HL-7702 cells. In addition, various biological assays show that Ru1 and Ru6, especially the Ru1 complex, are telomerase inhibitors targeting c-myc G4 DNA and also cause apoptosis of Hep-G2 cells. With the same Ru center, the in vitro antitumor activity and cellular uptake ability of the 4-EtN-Phtpy and 1-Bip-Phtpy ligands follow the order 4-EtN-Phtpy > 1-Bip-Phtpy.

Facile preparation of core cross-linked nanomicelles based on graft copolymers with pH responsivity and reduction sensitivity for doxorubicin delivery

To achieve passive targeting and controlled drug release at tumor sites trigged by low pH value and high level of glutathione (GSH), optimized delivery system for doxorubicin (DOX) based on core cross-linked nanomicelles was developed in this research. Particularly, methoxypoly(ethylene glycol)-nitrophenyl carbonate (mPEG-NPC) and 3,4-dihydroxyphenylaceticacid were grafted onto synthesized poly(N,N′-cystamine bisacrylamide-N-Boc-1,2-diaminoethane) (poly(CBA-DAE)) to give methoxypoly(ethylene glycol)-g-poly(N,N′-cystamine bisacrylamide-N-Boc-1, 2-diaminoethane)-g-3, 4-dihydroxyphenylaceticacid (mPEG-g-SS-PCD-DA). Core cross-linked micelles (CCLMs/SS) with a decreased average particle size of 121nm were prepared by adding Fe3+ into uncross-linked micelles (UCLMs/SS) self-assembled from mPEG-g-SS-PCD-DA. DOX-loaded CCLMs/SS exhibited minimal drug leakage (17.3%) under simulated blood conditions compared to DOX-loaded UCLMs/SS (31.3%). Fast drug release (52.4%) of DOX-loaded CCLMs/SS was achieved compared to DOX-loaded CCLMs/CC (32.9%) without disulfide bonds under simulated lysosomes condition over 42h. The cytotoxicity of DOX-loaded CCLMs/SS against A549 cells pretreated with 40mM NH4Cl was decreased significantly compared to that without NH4Cl treatment, and it is higher than that of DOX-loaded CCLMs/CC, further confirmed DOX release was triggered by the low pH value and high level of reductive agents of lysosomes. Compared with free DOX, DOX-loaded CCLMs/SS showed enhanced cellular uptake ability during 24h of incubation through endocytosis. Besides, charge conversion of micelles happened when pH varied from 7.4 to 6.5, which facilitates the cellular uptake against A549 cells. In summary, all these results indicated that CCLMs/SS as a new type of intelligent nanocarriers exhibited excellent potential for drug delivery.

Nanomicelle drug with acid-triggered doxorubicin release and enhanced cellular uptake ability based on mPEG-graft-poly(N-(2-aminoethyl)-L-aspartamide)-hexahydrophthalic acid copolymers.

In order to achieve the passive tumor targeting and acid-triggered drugs release in lysosomes, optimized delivery system for doxorubicin based on pH-sensitive complex nanomicelles with suitable particle size was developed in this research. Particularly, poly(L-succinimide) was thoroughly ring-opened by ethylenediamine to give the poly(N-(2-aminoethyl)-L-aspartamide). Then, graft copolymer mPEG-graft-poly(N-(2-aminoethyl)-L-aspartamide)-hexahydrophthalic acid (mPEG-g-P(ae-Asp)-Hap) was synthesized by grafting mPEG-2000 and hexahydrophthalic anhydride onto poly(N-(2-aminoethyl)-L-aspartamide). Invitro studies revealed that mPEG-g-P(ae-Asp)-Hap copolymer was stable in neutral solutions but tend to be hydrolyzed under acidic condition, which was attributed to the acid-sensitive properties of hexahydrophthalic amides (β-carboxylic amides). MPEG-g-P(ae-Asp)-Hap copolymer with critical aggregation concentration of 0.166 mg·mL-1 could self-assemble into stable blank nanomicelles with an average particle hydrodynamic diameter of 98.1 nm, but the hydrodynamic diameter of doxorubicin-loaded nanomicelles (mPEG-g-P(ae-Asp)-Hap·Dox) was smaller and approximately 77.5 nm. MPEG-g-P(ae-Asp)-Hap·Dox nanomicelles showed sustained drug release profiles over 34 h, and the cumulative drug release showed a tendency to increase from 25% to 62% with the pH value decreasing from 7.4 to 5.0 due to the acid-triggered disassembly of nanomicelles. The cytotoxicity of mPEG-g-P(ae-Asp)-Hap·Dox nanomicelles against A549 treated with 40 mM NH4Cl (lysosomotropic weak bases) was decreased significantly than that without NH4Cl treatment, further confirmed the drug release from the nanomicelles was triggered by the low pH value of lysosome (pH 5.0). Compared with doxorubicin HCl, mPEG-g-P(ae-Asp)-Hap·Dox nanomicelle drug showed enhanced cellular uptake ability during 2 or 4 h of incubation due to the endocytosis mechanism of nanomicelle drug. In summary, the cleavage of pH-sensitive β-carboxylic amides bonds on the hydrophobic branch of mPEG-g-P(ae-Asp)-Hap copolymer triggered the disassembly of the nanomicelles and release of doxorubicin in the acidic lysosomal compartments of cancer cells. These nanomicelles exhibited excellent potential for drug delivery due to their smart properties-PEGylation, suitable size, and acid-triggered drug release.

Reduction-Triggered Release of CPT from Acid-Degradable Polymeric Prodrug Micelles Bearing Boronate Ester Bonds with Enhanced Cellular Uptake.

The aim of this research is to develop a novel type of camptothecin (CPT) prodrug micelles bearing boronate ester bonds as a smart nanosystem with enhanced cellular uptake and controlled drug release based on diblock copolymer abbreviated as PEG-BC-PGlu-ss-CPT. Particularly, boronate ester bond was introduced to achieve acid-triggered de-PEGylation and succeeding boronic acid-mediated enhanced cellular uptake. Besides, CPT was conjugated to the prodrug monomer through a disulfide bond to realize reduction-responsive drug release. The resultant copolymer PEG-BC-PGlu-ss-CPT could self-assemble into spherical nanomicelles in water. The degradation half-life time of PEG-BC-PGlu-ss-CPT copolymer decreased sharply from 96.27 h to only 5.7 h with pH value decreasing from 7.4 to 5.0, indicating the acid-degradable potential, which corresponded to size change monitoring. The cumulative CPT release from prodrug micelles increased significantly from 8.5 ± 1.73 to 82.9 ± 2.29% with an increase of dithiothreitol (DTT) concentration from 20 μM to 10 mM at pH 7.4, illustrating the reduction-responsive drug release property of prodrug micelles. The half maximal inhibitory concentration (IC50) value of prodrug micelles against HepG2 cells decreased from 1.06 to 0.68 μg/mL with the decrement of pH value from 7.4 to 6.0, proving that the utilization of boronate ester bonds was beneficial for enhancing antiproliferative activity. Interestingly, prodrug micelles exhibited enhanced cellular uptake ability against HepG2 cells compared to that of HL7702 cells, further confirming boronic acid-mediated enhanced endocytosis. In brief, this novel type of intelligent prodrug micelles possessed great potential as a smart nanosystem for antitumor drug delivery.