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This report provides an overview of the discussions, presentations, and consensus thinking from the Workshop on Smart Data Collection for CryoEM held at the New York Structural Biology Center on April 6-7, 2022. The goal of the workshop was to address next generation data collection strategies that integrate machine learning and real-time processing into the workflow to reduce or eliminate the need for operator intervention.

Single-particle cryo-electron microscopy (cryoEM) is a swiftly growing method for understanding protein structure. With increasing demand for high-throughput, high-resolution cryoEM services comes greater demand for rapid and automated cryoEM grid and sample screening. During screening, optimal grids and sample conditions are identified for subsequent high-resolution data collection. Screening is a major bottleneck for new cryoEM projects because grids must be optimized for several factors, including grid type, grid hole size, sample concentration, buffer conditions, ice thickness and particle behavior. Even for mature projects, multiple grids are commonly screened to select a subset for high-resolution data collection. Here, machine learning and novel purpose-built image-processing and microscope-handling algorithms are incorporated into the automated data-collection software Leginon, to provide an open-source solution for fully automated high-throughput grid screening. This new version, broadly called Smart Leginon, emulates the actions of an operator in identifying areas on the grid to explore as potentially useful for data collection. Smart Leginon Autoscreen sequentially loads and examines grids from an automated specimen-exchange system to provide completely unattended grid screening across a set of grids. Comparisons between a multi-grid autoscreen session and conventional manual screening by 5 expert microscope operators are presented. On average, Autoscreen reduces operator time from ∼6 h to Smart Leginon to target holes that are particularly difficult to identify is analyzed. Finally, the utility of Smart Leginon is illustrated with three real-world multi-grid user screening/collection sessions, demonstrating the efficiency and flexibility of the software package. The fully automated functionality of Smart Leginon significantly reduces the burden on operator screening time, improves the throughput of screening and recovers idle microscope time, thereby improving availability of cryoEM services.

Single particle cryo-electron microscopy (cryoEM) is a swiftly growing method for understanding protein structure. With increasing demand for high-throughput, high-resolution cryoEM services comes greater demand for rapid and automated cryoEM grid and sample screening. During screening, optimal grids and sample conditions are identified for subsequent high-resolution data collection. Screening is a major bottleneck for new cryoEM projects because grids must be optimized over several factors, including grid type, grid hole size, sample concentration, buffer conditions, ice thickness, and particle behaviors. Even for mature projects, multiple grids are commonly screened to select a subset for high-resolution data collection. Here, machine learning and novel, purpose-built image processing and microscope-handling algorithms are incorporated into the automated data collection software, Leginon, to provide an open-source solution for fully automated, high-throughput grid screening. This new version, broadly called Smart Leginon, emulates the actions of an operator in identifying areas on the grid to explore as potentially useful for data collection. Smart Leginon Autoscreen sequentially loads and examines grids from an automated specimen exchange system to provide completely unattended grid screening across a set of grids. Comparisons between a multi-grid Autoscreen session and conventional manual screening by five expert microscope operators are presented. On average, Autoscreen reduces operator time from ∼6 hours to

In this work, we demonstrate the formation of supramolecular architectures from the assembly of single-tail single stranded DNA (ssDNA)-amphiphiles. Short ssDNA sequences of 10 nucleotides that were either unstructured or formed G-quadruplex secondary structures were conjugated to a single 4-(hexadecyloxy)benzamide tail, either directly or through a polycarbon (C

Background: It is plausible that interleukin polymorphisms may affect predisposition of autoimmune disorders such as systemic lupus erythematosus (SLE), but the results of so far published studies remain controversial. Objectives: The authors conducted this meta-analysis to clarify relationships between interleukin-1 (IL-1)/interleukin-4 (IL-4)/interleukin-6 (IL-6)/interleukin-10 (IL-10) polymorphisms and SLE by pooling the findings of eligible studies. Methods: A comprehensive search of PubMed, EMBASE, Web of Science, and CNKI was endorsed by the authors to identify already published studies. Fifty-seven studies were found to be eligible for meta-analyses. Results: The overall pooled meta-analyses yielded positive findings for IL-1A −889 C/T, IL-1B −31 T/C, IL-6 −174 G/C, IL-4 −590 C/T, and IL-10 −1,082 A/G polymorphisms. In addition, we also detected similar positive findings for IL-1B −511 C/T, IL-4 −590 C/T, IL-10 −592 A/C, IL-10 −819 C/T, and IL-10 −1,082 A/G polymorphisms in Asians, and such positive findings were also observed for IL-1A −889 C/T, IL-6 −174 G/C, and IL-10 −1,082 A/G polymorphisms in Caucasians. Conclusions: The meta-analyses’ results suggest that IL-1A −889 C/T, IL-1B −31 T/C, IL-6 −174 G/C, IL-4 −590 C/T, and IL-10 −1,082 A/G polymorphisms might affect predisposition of SLE.

Cryo–electron microscopy (cryo-EM) continues its remarkable growth as a method for visualizing biological objects, which has been driven by advances across the entire pipeline. Developments in both single-particle analysis and in situ tomography have enabled more structures to be imaged and determined to better resolutions, at faster speeds, and with more scientists having improved access. This review highlights recent advances at each stageof the cryo-EM pipeline and provides examples of how these techniques have been used to investigate real-world problems, including antibody development against the SARS-CoV-2 spike during the recent COVID-19 pandemic.

Description, DNA nanotubes can cross blood-brain tumor barrier and enhance survival with no toxicity when used for the local delivery of DOX., Effective treatment of glioblastoma remains a daunting challenge. One of the major hurdles in the development of therapeutics is their inability to cross the blood-brain tumor barrier (BBTB). Local delivery is an alternative approach that can still suffer from toxicity in the absence of target selectivity. Here, we show that nanotubes formed from self-assembly of ssDNA-amphiphiles are stable in serum and nucleases. After bilateral brain injections, nanotubes show preferential retention by tumors compared to normal brain and are taken up by glioblastoma cells through scavenger receptor binding and macropinocytosis. After intravenous injection, they cross the BBTB and internalize in glioblastoma cells. In a minimal residual disease model, local delivery of doxorubicin showed signs of toxicity in the spleen and liver. In contrast, delivery of doxorubicin by the nanotubes resulted in no systemic toxicity and enhanced mouse survival. Our results demonstrate that ssDNA nanotubes are a promising drug delivery vehicle to glioblastoma.

Burkholderia pseudomallei is a Gram-negative soil-dwelling bacillus that causes melioidosis, a frequently fatal infectious disease, in tropical and subtropical regions. Previous studies have identified the overall genetic and evolutionary characteristics of B. pseudomallei on a global scale, including its origin and transmission routes. However, beyond its known hyperendemicity foci in northern Australia and Southeast Asia, the distribution and genetic characteristics of B. pseudomallei in most tropical regions remain poorly understood, including in southern China. Here, we sequenced the genomes of 122 B. pseudomallei strains collected from Hainan, an island in southern China, in 2002–2018, to investigate the population structure, relationships with global strains, local epidemiology, and virulence and antimicrobial-resistance factors. A phylogenetic analysis and hierarchical clustering divided the Hainan strains into nine phylogenic groups (PGs), 80 % of which were concentrated within five major groups (group 1: corresponding to minor sequence types [STs], 12.3 %; group 3: ST46 and ST50, 31.1 %; group 9: ST58, 13.1 %; group 11: ST55, 8.2 %; group 15: mainly ST658, 15.6%). A phylogenetic analysis that included global strains suggested that B. pseudomallei in Hainan originated from Southeast Asian countries, transmitted in multiple historical importation events. We also identified several mutual transmission events between Hainan and Southeast Asian countries in recent years, including three importation events from Thailand and Singapore to Hainan and three exportation events from Hainan to Singapore, Malaysia, and Taiwan island. A statistical analysis of the temporal distribution showed that the Hainan strains of groups 3, 9, and 15 have dominated the disease epidemic locally in the last 5 years. The spatial distribution of the Hainan strains demonstrated that some PGs are distributed in different cities on Hainan island, and by combining phylogenic and geographic distribution information, we detected 21 between-city transmission events, indicating its frequent local transmission. The detection of virulence factor genes showed that 56 % of the Hainan strains in group 1 encode a B. pseudomallei -specific adherence factor, boaB, confirming the specific pathogenic characteristics of the Hainan strains in group 1. An analysis of the antimicrobial-resistance potential of B. pseudomallei showed that various kinds of alterations were identified in clinically relevant antibiotic resistance factors, such as AmrR, PenA and PBP3, etc. Our results clarify the population structure, local epidemiology, and pathogenic characteristics of B. pseudomallei in Hainan, providing further insight into its regional and global transmission networks and improving our knowledge of its global phylogeography.

A DNA-based artificial metalloenzyme (ArM) consisting of copper (II) complex of 4,4’-dimethyl-2,2’-bipyridine (dmbipy-Cu) bound to double-stranded DNA (dsDNA) as short as 8 base pairs with only two contiguous central G•C pairs (G for guanine and C for cytosine), catalyzes highly enantioselective Diels-Alder reaction in water. Molecular simulations indicate that these minimal sequences provide a single site where dmbipy-Cu is groove-bound and able to function as an enantioselective catalyst. Enantioselective preference inverses when D-DNA is replaced with L-DNA. When the DNA is conjugated to a hydrophobic tail, the obtained ArMs exhibit enantioselective performance in a methanol-water mixture superior to that of non-amphiphilic dsDNA, and dsDNA-amphiphiles with more complex G•C-rich sequences.

An aptamer-amphiphile was designed that binds to β-lactoglobulin (β-LG), a major allergen from cow's milk. For this work, a 23-nucleotide ssDNA aptamer β-LG-23, capable of forming antiparallel G-quadruplexes was used, and its specificity and binding affinity of 22 ± 2 nM for β-LG were evaluated via enzyme-linked apta-sorbent assay (ELASA). The β-LG-23 aptamer was synthesized as an amphiphile by conjugating it to a C16 double tail via different spacers, and the effect of the spacers on the binding affinity and secondary structure of the aptamer was investigated. From all amphiphiles tested, direct conjugation of the aptamer to the tail gave the lowest binding affinity to β-LG (37 ± 2 nM), while maintaining the antiparallel G-quadruplex secondary structure of the aptamer. As a proof of concept, the β-LG-23 aptamer-amphiphile was used to decorate the interface of a liquid crystal (LC) and effectively detected 10 nM or 0.18 ppm of β-LG with a 20 min equilibration time, thus demonstrating that it has the potential to be used for fast and label-free detection of β-LG.

Structural maintenance of chromosomes (SMC) complexes are critical chromatin modulators. In eukaryotes, the cohesin and condensin SMC complexes organize chromatin, while the Smc5/6 complex directly regulates DNA replication and repair. The molecular basis for the distinct functions of Smc5/6 is poorly understood. Here, we report an integrative structural study of the budding yeast Smc5/6 holo-complex using electron microscopy, cross-linking mass spectrometry, and computational modeling. We show that the Smc5/6 complex possesses several unique features, while sharing some architectural characteristics with other SMC complexes. In contrast to arm-folded structures of cohesin and condensin, Smc5 and Smc6 arm regions do not fold back on themselves. Instead, these long filamentous regions interact with subunits uniquely acquired by the Smc5/6 complex, namely the Nse2 SUMO ligase and the Nse5/Nse6 subcomplex, with the latter also serving as a linchpin connecting distal parts of the complex. Our 3.0-Å resolution cryoelectron microscopy structure of the Nse5/Nse6 core further reveals a clasped-hand topology and a dimeric interface important for cell growth. Finally, we provide evidence that Nse5/Nse6 uses its SUMO-binding motifs to contribute to Nse2-mediated sumoylation. Collectively, our integrative study identifies distinct structural features of the Smc5/6 complex and functional cooperation among its coevolved unique subunits.

Despite decades of research, there are few targeted treatment options available for triple negative breast cancer (TNBC), leaving chemotherapy, and radiation treatment regimes with poor response and high toxicity. Herein aptamer‐amphiphiles were synthesized which selectively bind to the mucin‐1 (MUC1) glycoprotein that is overexpressed in TNBC cells. These amphiphiles have a fluorescent tail (1,8‐naphthalimide or 4‐nitro‐1,8‐naphthalimide) which enables self‐assembly of the amphiphiles and allows for easy visualization without the requirement for further conjugation of a fluorophore. Interestingly, the length of the alkyl spacer (C4 or C12) between the aptamer and tail was shown to influence the morphology of the self‐assembled structure, and thus its ability to internalize into the TNBC cells. While both the MUC1 aptamer‐C4‐napthalimide spherical micelles and the MUC1 aptamer‐C12‐napthalimide long cylindrical micelles showed internalization into MDA‐MB‐468 TNBC cells but not the noncancerous MCF‐10A breast cells, the cylindrical micelles showed greatly enhanced internalization into the MDA‐MB‐468 cells. Similar patterns of enhanced binding and internalization were observed between the MUC1 aptamer‐C12‐napthalimide cylindrical micelles and SUM159 and MDA‐MB‐231 TNBC cells. The MUC1 aptamer cylindrical micelles were not toxic to the cells, and when used to deliver doxorubicin to the TNBC cells, were shown to be as cytotoxic as free doxorubicin. Moreover, a pharmacokinetic study in mice showed a prolonged systemic circulation time of the MUC1 aptamer cylindrical micelles. There was a 4.6‐fold increase in the elimination half‐life of the aptamer cylindrical micelles, and their clearance decreased 10‐fold compared to the MUC1 aptamer spherical micelles. Thus, the MUC1 aptamer‐C12‐napthalimide nanofibers represent a promising vehicle that could be used for easy visualization and targeted delivery of therapeutic loads to TNBC cells.

Injectable and biodegradable supramolecular hydrogels were prepared by nucleobase (adenine/thymine)-terminated poly(ethylene oxide)s (A-PEG-A/T-PEG-T) and α-cyclodextrin (α-CD). The supramolecular hydrogels were thoroughly characterized by WXRD, rheometer, and SEM. The gelation time depended on the molecular weight of PEG and the concentration of polymer precursors. The rheological studies showed enhanced elastic modulus (G′) of hydrogels, because of the hydrogen-bonding between A and T acting as additional network junctions. In vitro evaluation showed that the supramolecular hydrogels have acceptable biocompatibility, and are suitable for sustained and controlled release of loaded antitumor drugs. Gel formation was also confirmed when the supramolecular hydrogels were subcutaneously injected into rats. In addition, in vivo experiments employing U14 cancer cell xenograft-bearing mice showed that the intratumoral injection of a DOX-loaded A-PEG-A/T-PEG-T/α-CD gel inhibited tumor growth more effectively than that of free DOX, DOX-loaded PEG/α-CD gel, saline or gel alone. Hence, such a simple and convenient anti-cancer drug delivery system of a A-PEG-A/T-PEG-T/α-CD supramolecular hydrogel would be a promising candidate for many biomedical applications, especially in the area of the chemotherapy of solid tumors.

In this work we hypothesized that the chemokine fractalkine can serve as a cancer molecular target. We engineered aptamer micelles functionalized with an outer poly(ethylene glycol) (PEG) corona, and investigated the extent and efficacy of using them as a targeting tool against fractalkine-expressing colon adenocarcinoma cells. In vitro cell binding results showed that aptamer micelles bound and internalized to fractalkine-expressing cancer cells with the majority of the micelles found free in the cytoplasm. Minimal surface binding was observed by healthy cells. Even though partial PEGylation did not prevent serum adsorption, micelles were highly resistant to endonuclease and exonuclease degradation. In vivo biodistribution studies and confocal studies demonstrated that even though both aptamer and control micelles showed tumor accumulation, only the aptamer micelles internalized into fractalkine-expressing cancer cells, thus demonstrating the potential of the approach and showing that fractalkine may serve as a specific target for nanoparticle delivery to cancer cells.

A double pH-responsive supramolecular copolymer micelle was successfully developed, which was based on complementary multiple hydrogen bonds of nucleobases and acetalated dextran. The thymine-terminated poly(ethylene glycol) (T-PEG-T) and adenine-terminated acetalated dextran (AcDEX-A) were synthesized and used to construct supramolecular amphiphilic triblock copolymer AcDEX-A:T-PEG-T:A-AcDEX, which could be further self-assembled into supramolecular micelles in water. These micelles were stable at pH 7.4, but disruptive at pH 5.0 due to the double pH-sensitivity of hydrogen bonds within the copolymer backbones and the hydrolysis of the acetalated dextran. The hydroxyl coverage of Ac-DEX had an important effect on the critical micelle concentration (CMC) and particle size of AcDEX-A:T-PEG-T:A-AcDEX micelles, which were observed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). In addition, turbidometry was utilized to evaluate the effect of pH-responsivity of AcDEX-A:T-PEG-T:A-AcDEX micelles and these supramolecular micelles were completely decomposed at pH 5.0. Cytotoxicity evaluation showed good biocompatibility of these micelles, and doxorubicin (DOX) was encapsulated into supramolecular micelles as a model drug. The in vitro drug release profile showed that the DOX release speed at pH 5.0 could be adjusted by changing the hydroxyl coverage of Ac-DEX. Meanwhile, DOX-loaded supramolecular micelles could be efficiently internalized into cancer cells and showed similar inhibition of proliferation of the Hela cell as free DOX. This work provided a new method for enabling a rapid intracellular release of drugs by using double pH-sensitive hydrogen bonds and acetalated dextran, which would have the potential to be applied in controlled drug delivery.

A pH-sensitive drug release system using acetalated-dextran as valves was designed to manipulate smart intracellular release of anticancer drugs. Dextran was grafted onto the exterior of MSN through a click reaction, and followed by acetalation to generate the final carriers of MSN–Dex-Ac. The hydrophobic Dex-Ac would act as valves on the MSN surface to block the entrapped drugs inside the MSN pores. While under acidic conditions mimicking the micro-environment of endosomal/lysosomal compartments, the valves could be opened by acetal hydrolysis to recover the acetalated-dextran to its hydrophilic state, resulting in fast drug release. In vitro drug release profile clearly showed that DOX release was restricted at pH 7.4 by the valves, while it was accelerated under acidic conditions. Fast endocytosis and intracellular DOX release was observed by confocal laser scanning microscopy (CLSM). Cytotoxicity evaluation showed good biocompatibility with the carriers. In vitro MTT assays revealed that the DOX-loaded particles exhibited comparable antitumor activity with free DOX towards HeLa cells.

A comprehensive study is reported on the effect of salt concentration, polyelectrolyte block length, and polymer concentration on the morphology and structural properties of nanoaggregates self-assembled from BAB single-strand DNA (ssDNA) triblock polynucleotides in which A represents polyelectrolyte blocks and B represents hydrophobic neutral blocks. A morphological phase diagram above the gelation point is developed as a function of solvent ionic strength and polyelectrolyte block length utilizing an implicit solvent ionic strength method for dissipative particle dynamics simulations. As the solvent ionic strength increases, the self-assembled DNA network structures shrinks considerably, leading to a morphological transition from a micellar network to worm-like or hamburger-shape aggregates. This study provides insight into the network morphology and its changes by calculating the aggregation number, number of hydrophobic cores, and percentage of bridge chains in the network. The simulation results are corroborated through cryogenic transmission electron microscopy on the example of the self-assembly of ssDNA triblocks.

Mesoporous silica nanoparticles (MSN) with 1,2-bidentate carboxyl groups on the surface reacted with 1,2-diaminecyclohexano platinum(II) dinitrate (DACH-Pt-(NO3)2) which is an active anticancer species of clinic relevant oxaliplatin to form MSN-Pt. The modification of the parent particles was monitored by (13)C, (29)Si solid-state NMR, X-ray measurements (XRD) and Fourier transform infrared spectroscopy (FT-IR). After loading with platinum drugs, MSN-Pt exhibited two strong Pt4f signals as indicated by X-ray photoelectron spectroscopy (XPS). The platinum content in the conjugates was calculated to be 9.7% according to ICP-MS measurements. Confocal laser scanning microscopy (CLSM) displayed that MSN-Pt were uptaken fast by HepG-2 cells and concentrated within endosomes and lysosomes. In vitro MTT assay of MSN-Pt demonstrated an improved cytotoxicity against HepG-2 cells than that of free oxaliplatin. This is due to the fact that MSN-Pt expressed higher platinum intracellular uptake and more DNA binding (Pt-DNA adducts) than free oxaliplatin. Hence this work highlighted that the platinum loaded MSN nanoparticles could be a promising future intelligent drug delivery system.

A photo-cross-linked micelle is synthesized via photodimerization of thymine moieties fabricated from amphiphilic block copolymers (mPEG-b-P(LA-co-MPT). The crosslinking behavior is monitored by UV-Vis spectra and H-1 NMR. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) showed that cross-linked micelles had smaller sizes than their uncross-linked precursors. In vitro studies reveal that cross-linking of the micelle cores results in a slow drug release and faster cellular uptake in comparison with uncross-linked ones in MCF-7 and Hela cells. Moreover, the paclitaxel (PTX)-loaded core-cross-linked micelles exhibit similar anticancer efficacy as free PTX. This work provides a convenient tool for designing a more stable structure in the blood circulation to realize a controlled drug delivery.

Stimuli-responsive micellar nanoparticles are gaining considerable attention in the field of drug delivery because of their great advantages of high efficiency in tumor accumulation through the enhanced permeability and retention effect and rapid drug release. In this study, a novel catechol moiety-containing amphiphilic, biodegradable polymeric carrier, monomethoxy poly(ethylene glycol)-block-poly(e-caprolactone)-block-poly(L-lysine)-graft-[3-(3,4-dihydroxyphenyl)propionic acid], was employed for delivery of the anticancer drug bortezomib (BTZ) to cancer cells. The strategy is based on the facile conjugation of BTZ to catechol-containing polymeric carriers through boronate formation, which could dissociate in acidic environments to liberate its payload BTZ to inhibit proteasome function. This reducing BTZ–catechol interaction with decreasing pH value was demonstrated by microcalorimetry analysis using isothermal titration calorimetry. Notably, the obtained micellar BTZ complex that self-assembled from drug-loaded amphiphilic polymer was internalized effectively by MCF-7 breast cancer cells and resulted in significant 26S proteasome inhibition. Furthermore, the micellar BTZ complex induced remarkable apoptosis on MCF-7 and HeLa cancer cells and exhibited little toxicity on HEK293 normal cells. More importantly, systemic delivery of micellar BTZ complex prolonged its blood circulation and resulted in significant accumulation in the tumor site relative to free drug, thus suggesting therapeutic promise for micellar BTZ delivery in cancer therapy.

Mesoporous silica nanoparticles (MSN)–polymer hybrid combined with the aliphatic biodegradable polyester caps on the surface were first developed in order to manipulate the smart intracellular release of anticancer drugs. First, poly(ethylene glycol)-b-poly(e-caprolactone) (PEG-PCL) was successfully grafted on the surface of MSN via disulfide bonds which could cleave under a reduction environment in tumor cells. The anticancer drug doxorubicin (DOX) was encapsulated into the particle pores. The in vitro drug release profile showed that DOX release was significantly restricted by the polymer caps at pH 7.4, while it was greatly accelerated upon the addition of GSH. Cytotoxicity evaluation showed good biocompatibility with the hybrid particles. Fast endocytosis and intracellular DOX release were observed by confocal laser scanning microscopy (CLSM). The DOX-loaded particles exhibited comparable antitumor activity with free DOX towards HeLa cells and showed in a time-dependent manner. This work developed an extensive method of utilizing aliphatic biodegradable polyesters as polymer caps for MSN to control drug delivery. The paper might offer a potential option for cancer therapy.

A photo-cross-linked micelle is synthesized via photodimerization of thymine moieties fabricated from amphiphilic block copolymers (mPEG-b-P(LA-co-MPT). The crosslinking behavior is monitored by UV-Vis spectra and (1) H NMR. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) showed that cross-linked micelles had smaller sizes than their uncross-linked precursors. In vitro studies reveal that cross-linking of the micelle cores results in a slow drug release and faster cellular uptake in comparison with uncross-linked ones in MCF-7 and Hela cells. Moreover, the paclitaxel (PTX)-loaded core-cross-linked micelles exhibit similar anticancer efficacy as free PTX. This work provides a convenient tool for designing a more stable structure in the blood circulation to realize a controlled drug delivery.

A novel biodegradable pseudo-poly(aminoacid) copolymer (mPEG–HRSCP–mPEG) was produced via a mechanism of step polymerization through Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) (click polymerization) using dialkyned-cystine and diazide monomers. The disulfide and hydroxyl groups were repeatedly arranged in the polymer backbone. The introduced hydroxyl groups could not only adjust the hydrophilicity of the amphiphilic polymer, but also improve the DOX loading efficiency and solubility. Compared with the unmodified one, the polymer containing hydroxyl groups has increased hydrophilicity with a larger critical aggregation concentration (CAC) value, lower water contact angle, higher drug loading content and drug loading efficiency. The pH and reduction sensitivities of this drug delivery system (DDS) were investigated using Dynamic Light Scattering (DLS) to monitor the changes of average diameters. DOX was loaded as a model drug and in vitro release experiments demonstrated that DOX release was accelerated by an addition of 10 mM glutathione (GSH) or under acidic conditions rather than under physiological conditions. Our study on in vitro anticancer efficiency showed that DOX-loaded nanoparticles had a higher cytotoxicity towards GSH pretreated HeLa cells. These newly designed copolymer nanoparticles can be used as novel and impactful pH and reduction dual-responsive nanocarriers for intelligent DOX delivery.

Efficient delivery of anti-cancer drugs into tumor cells for enhancing the intracellular drug concentration is a major challenge for cancer therapy due to the instability of drug-loading vehicle. In this report, we developed a simple method to stabilize the nanostructure of micelles only by bubbling air to crosslink the outer layer of the micelle core. Dopamine was conjugated to a biodegradable triblock copolymer monomethoxy poly(ethylene glycol)-b-poly(2-methyl-2-carboxyl-propylene carbonate)-b-poly(L-lactide) (mPEG-b-PMCC-b-PLA) to obtain dopamine grafted copolymer mPEG-b-P(MCC-g-dopamine)-b-PLA. After self-assembly, the core cross-linked micelles were then prepared by the oxidative self-polymerization of dopamine in the middle hydrophobic phase of the micelles. The cross-linked micelles had smaller sizes and narrower particle size distributions than their uncross-linked precursors. The improved stability was confirmed by critical micelle concentration (CMC) experiments and 1H NMR spectra. The kinetics and processes of oxidative cross-linking of micelles under air flux were monitored by UV-Vis spectroscopy and transmission electron microscopy (TEM). These core cross-linked micelles were able to load doxorubicin (DOX) with superior loading capacity of up to 19.5% (w/w, drug/micelle) with high drug loading efficiency (97.5%). Compared with the uncross-linked ones, drug release efficacy from the cross-linked micelles extremely decreased at pH 7.4. However, a properly sustained release occurred at pH 5.0, which is very favorable for drug delivery in tumor cells. The DOX-loaded micelles had similar cytotoxicity as the free drug and could be effectively internalized into MDA-MB-231 cells. This controllable and convenient approach for preparing core cross-linked micelles will have a pragmatic future in stabilizing the architecture of nanocarriers for drug delivery.