Xianglong Hu

Polyprodrug Amphiphiles: Hierarchical Assemblies for Shape-Regulated Cellular Internalization, Trafficking, and Drug Delivery

Solution self-assembly of block copolymers (BCPs) typically generates spheres, rods, and vesicles. The reproducible bottom-up fabrication of stable planar nanostructures remains elusive due to their tendency to bend into closed bilayers. This morphological vacancy renders the study of shape effects on BCP nanocarrier-cell interactions incomplete. Furthermore, the fabrication of single BCP assemblies with built-in drug delivery functions and geometry-optimized performance remains a major challenge. We demonstrate that PEG-b-PCPTM polyprodrug amphiphiles, where PEG is poly(ethylene glycol) and PCPTM is polymerized block of reduction-cleavable camptothecin (CPT) prodrug monomer, with >50 wt % CPT loading content can self-assemble into four types of uniform nanostructures including spheres, large compound vesicles, smooth disks, and unprecedented staggered lamellae with spiked periphery. Staggered lamellae outperform the other three nanostructure types, exhibiting extended blood circulation duration, the fastest cellular uptake, and unique internalization pathways. We also explore shape-modulated CPT release kinetics, nanostructure degradation, and in vitro cytotoxicities. The controlled hierarchical organization of polyprodrug amphiphiles and shape-tunable biological performance opens up new horizons for exploring next-generation BCP-based drug delivery systems with improved efficacy.

Amphiphilic multiarm star block copolymer-based multifunctional unimolecular micelles for cancer targeted drug delivery and MR imaging.

We report on the fabrication of multifunctional polymeric unimolecular micelles as an integrated platform for cancer targeted drug delivery and magnetic resonance imaging (MRI) contrast enhancement under in vitro and in vivo conditions. Starting from a fractionated fourth-generation hyperbranched polyester (Boltorn H40), the ring-opening polymerization of ɛ-caprolactone (CL) from the periphery of H40 and subsequent terminal group esterification with 2-bromoisobutyryl bromide afforded star copolymer-based atom transfer radical polymerization (ATRP) macroinitiator, H40-PCL-Br. Well-defined multiarm star block copolymers, H40-PCL-b-P(OEGMA-co-AzPMA), were then synthesized by the ATRP of oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA) and 3-azidopropyl methacrylate (AzPMA). This was followed by the click reaction of H40-PCL-b-P(OEGMA-co-AzPMA) with alkynyl-functionalized cancer cell-targeting moieties, alkynyl-folate, and T(1)-type MRI contrast agents, alkynyl-DOTA-Gd (DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakisacetic acid), affording H40-PCL-b-P(OEGMA-Gd-FA). In aqueous solution, the amphiphilic multiarm star block copolymer exists as structurally stable unimolecular micelles possessing a hyperbranched polyester core, a hydrophobic PCL inner layer, and a hydrophilic P(OEGMA-Gd-FA) outer corona. H40-PCL-b-P(OEGMA-Gd-FA) unimolecular micelles are capable of encapsulating paclitaxel, a well-known hydrophobic anticancer drug, with a loading content of 6.67 w/w% and exhibiting controlled release of up to 80% loaded drug over a time period of ∼120 h. In vitro MRI experiments demonstrated considerably enhanced T(1) relaxivity (18.14 s(-1) mM(-1)) for unimolecular micelles compared to 3.12 s(-1) mM(-1) for that of the small molecule counterpart, alkynyl-DOTA-Gd. Further experiments of in vivo MR imaging in rats revealed good accumulation of unimolecular micelles within rat liver and kidney, prominent positive contrast enhancement, and relatively long duration of blood circulation. The reported unimolecular micelles-based structurally stable nanocarriers synergistically integrated with cancer targeted drug delivery and controlled release and MR imaging functions augur well for their potential applications as theranostic systems.

Cell-Penetrating Hyperbranched Polyprodrug Amphiphiles for Synergistic Reductive Milieu-Triggered Drug Release and Enhanced Magnetic Resonance Signals

The rational design of theranostic nanoparticles exhibiting synergistic turn-on of therapeutic potency and enhanced diagnostic imaging in response to tumor milieu is critical for efficient personalized cancer chemotherapy. We herein fabricate self-reporting theranostic drug nanocarriers based on hyperbranched polyprodrug amphiphiles (hPAs) consisting of hyperbranched cores conjugated with reduction-activatable camptothecin prodrugs and magnetic resonance (MR) imaging contrast agent (Gd complex), and hydrophilic coronas functionalized with guanidine residues. Upon cellular internalization, reductive milieu-actuated release of anticancer drug in the active form, activation of therapeutic efficacy (>70-fold enhancement in cytotoxicity), and turn-on of MR imaging (∼9.6-fold increase in T1 relaxivity) were simultaneously achieved in the simulated cytosol milieu. In addition, guanidine-decorated hPAs exhibited extended blood circulation with a half-life up to ∼9.8 h and excellent tumor cell penetration potency. The hyperbranched chain topology thus provides a novel theranostic polyprodrug platform for synergistic imaging/chemotherapy and enhanced tumor uptake.

Thiol and pH dual-responsive dynamic covalent shell cross-linked micelles for triggered release of chemotherapeutic drugs

We report on the fabrication of dynamic covalent shell cross-linked (SCL) micelles of amphiphilic diblock copolymers functionalized with aldehyde moieties in the hydrophilic block by utilizing difunctional crosslinkers cleavable in response to pH and thiols. Well-defined amphiphilic diblock copolymer, PCL-b-P(OEGMA-co-MAEBA), was synthesized via ring opening polymerization (ROP) of e-caprolactone (CL) and atom transfer radical polymerization (ATRP) of oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA) and p-(methacryloxyethoxy)benzaldehyde (MAEBA) comonomers. In aqueous solution, the diblock copolymer self-assembles into micelles consisting of hydrophobic PCL cores and hydrophilic P(OEGMA-co-MAEBA) coronas covalently anchored with aldehyde groups. The subsequent shell cross-linking reaction was conducted at pH 6.2 upon addition of difunctional dithiolbis(propanoic dihydrazide) (DTP). The formation of dynamic acylhydrazone cross-linking linkages was facilitated under the catalysis of aniline. The obtained SCL micelles can be de-crosslinked via two biologically relevant modes, namely, acidic pH-triggered cleavage of acylhydrazone bonds into aldehyde and hydrazide and thiol-triggered cleavage of disulfide linkages, which have been utilized for triggered release of physically encapsulated chemotherapeutic drugs. The dual-responsive dynamic covalent SCL micelles were examined by dynamic laser light scattering (LLS), 1H NMR, Ellmans assay, and enzymatic degradation tests. In addition, camptothecin (CPT)-loaded SCL micelles were used to investigate thiol and pH-modulated CPT release profiles. Compared with CPT-loaded non-cross-linked (NCL) micelles, CPT-loaded SCL micelles can largely minimize drug leakage under physiological conditions, whilst exhibiting accelerated drug release under mildly acidic or thiol-rich microenvironments, which are relevant to those of acidic organelles (endosomes and lysosomes) or cytosol within tumor cells. Cell cytotoxicity studies revealed that drug-free SCL micelles are almost nontoxic, whereas CPT-loaded SCL micelles can efficiently deliver chemotherapeutic drug (CPT) into HepG2 cells, leading to considerable nucleic accumulation at extended incubation duration. The reported dynamic covalent shell cross-linking strategy can exert intricate control concerning the micellar stability and the release profile of encapsulated drugs in response to biological microenvironments, which augurs well for their potential use as novel smart nanocarriers for drug delivery in cancer chemotherapy.

Ultrasensitive ratiometric fluorescent pH and temperature probes constructed from dye-labeled thermoresponsive double hydrophilic block copolymers

We report on the fabrication of highly sensitive ratiometric fluorescent pH and temperature probes based on thermoresponsive double hydrophilic block copolymers (DHBCs) with the two blocks labeled with two types of dyes possessing different pH-switchable emission characteristics. P(NIPAM-co-FITC)-b-P(OEGMA-co-RhBAM) DHBCs were synthesized via consecutive reversible addition–fragmentation chain transfer (RAFT) polymerizations in combination with post-modifications, where NIPAM, OEGMA, FITC, and RhBAM are N-isopropylacrylamide, oligo(ethylene glycol) monomethyl ether methacrylate, fluorescein isothiocyanate, and rhodamine B-based derivatives, respectively. Due to that FITC and RhBAM moieties exhibit prominent decrease and increase in emission intensities with decreasing solution pH, respectively, intensity ratios of characteristic RhBAM and FITC emission bands, I582/I522, of P(NIPAM-co-FITC)-b-P(OEGMA-co-RhBAM) unimers at 25 °C exhibit ∼39-fold changes in the range of pH 2–10. At elevated temperatures, thermo-induced formation of PNIPAM-core micelles enables effective fluorescence resonance energy transfer (FRET) between FITC and RhBAM moieties respectively located within micellar cores and coronas, and I582/I522 exhibits ∼52.5-fold changes in the same pH range. The reported dually modulated multicolor-emitting P(NIPAM-co-FITC)-b-P(OEGMA-co-RhBAM) DHBCs are capable of ultrasensitive fluorometric detection of solution pH and temperature in a ratiometric manner, which augurs well for their practical applications in sensing, imaging, and the fabrication of new generation of theranostic systems.

Mixed polymeric micelles as multifunctional scaffold for combined magnetic resonance imaging contrast enhancement and targeted chemotherapeutic drug delivery

We report on the utilization of mixed diblock copolymer micelles as an integrated multifunctional platform for the cancer cell-targeted delivery of chemotherapeutic drugs and magnetic resonance (MR) imaging contrast enhancement under in vitro and in vivo conditions. Two types of amphiphilic diblock copolymers, PCL-b-P(OEGMA-FA) and PCL-b-P(OEGMA-Gd), consisting of a hydrophobic poly(e-caprolactone) (PCL) block and a hydrophilic poly(oligo(ethylene glycol) monomethyl ether methacrylate) (POEGMA) block, covalently attached with folic acid (FA) and DOTA-Gd (Gd) moieties, respectively, were synthesized via the combination of atom transfer radical polymerization (ATRP), ring-opening polymerization (ROP), and “click” post-functionalization. Mixed micelles co-assembled from PCL-b-P(OEGMA-FA) and PCL-b-P(OEGMA-Gd) possess hydrophobic PCL cores for loading chemotherapeutic drugs and hydrophilic POEGMA outer coronas functionalized with FA and Gd complexes for synergistic functions of targeted delivery and MR imaging contrast enhancement. As-prepared nanosized mixed micelles are capable of physically encapsulating paclitaxel, a well-known hydrophobic anticancer drug, with a loading content of ∼5.0 w/w%, exhibiting controlled release of up to ∼60% loaded drugs over a duration of ∼130 h. In vitrocell viability assays revealed that drug-free mixed micelles are almost non-cytotoxic up to a concentration of 0.2 g L−1, whereas paclitaxel-loaded ones can effectively kill HeLa cells at the same concentration. In vitro MR imaging experiments indicated dramatically increased T1 relaxivity (26.29 s−1mM−1) for mixed micelles compared to that of small molecule counterpart, alkynyl-DOTA-Gd (3.12 s−1mM−1). Further in vivo MR imaging experiments in rabbits revealed considerably enhanced signal intensity, prominent positive contrast enhancement, improved accumulation and retention, and extended blood circulation duration for FA-labeled mixed micellar nanoparticles within the rabbit liver, as compared to those for FA-free mixed micelles and small molecule alkynyl-DOTA-Gd complex. These preliminary results indicate that the reported mixed micellar nanocarriers possess synergistically integrated functions of cancer-targeted drug delivery and controlled release, and MR imaging contrast enhancement, which augurs well for their potential application as a novel type of theranostic platform.

Polyion complex micellar nanoparticles for integrated fluorometric detection and bacteria inhibition in aqueous media.

The development of portable and inexpensive detection methods can significantly contribute to the prevention of water-borne infectious diseases caused by pathogenic bacteria. Here we designed a nanosystem capable of both bacterial detection and inhibition, where polyion complex (PIC) micelles are constructed from negatively-charged tetraphenylethylene (TPE) sulfonate derivatives, which exhibit the aggregation-induced emission (AIE) feature, and cationic diblock copolymers, poly(ethylene oxide)-b-quaternized poly(2-(dimethylamino)ethyl methacrylate) (PEO-b-PQDMA). Upon contacting with bacteria, the PIC nanosystem disintegrates presumably due to competitive binding of polycation blocks with negatively-charged bacterial surfaces. This process is accompanied by a conspicuous quenching of TPE fluorescence emission, serving as a real-time module for microbial detection. Furthermore, the sharp decrease in CFU is indicative of prominent anti-microbial activities. Thus, PIC micelles possess dual functions of fluorometric detection and inhibition for bacteria in aqueous media. By tuning the charge density of TPE sulfonate derivatives and chain length of cationic PQDMA blocks, optimal performance against Gram-negative Escherichia coli has been achieved with a detection limit of 5.5 × 10(4) CFU/mL and minimum inhibitory concentration (MIC) of 19.7 μg/mL. Tests against Gram-positive Staphylococcus aureus were also conducted to demonstrate versatility of the nanosystem.

Highly Stable Organic Small Molecular Nanoparticles as an Advanced and Biocompatible Phototheranostic Agent of Tumor in Living Mice

Near-infrared (NIR)-absorbing organic small molecules hold great promise as the phototheranostic agents for clinical translation by virtue of their intrinsic advantages such as well-defined chemical structure, high purity, and good reproducibility. However, most of the currently available ones face the challenges in varying degrees in terms of photothermal instability, and photobleaching/reactive oxygen nitrogen species (RONS) inresistance, which indeed impair their practical applications in precise diagnosis and treatment of diseases. Herein, we developed highly stable and biocompatible organic nanoparticles (ONPs) for effective phototheranostic application by design and synthesis of an organic small molecule (namely TPA-T-TQ) with intensive absorption in the NIR window. The TPA-T-TQ ONPs with no noticeable in vivo toxicity possess better capacities in photothermal conversion and photoacoustic imaging (PAI), as well as show far higher stabilities including thermal/photothermal stabilities, and photobleaching/RONS resistances, when compared with the clinically popularly used indocyanine green. Thanks to the combined merits, the ONPs can serve as an efficient probe for in vivo PAI in a high-contrast manner, which also significantly causes the stoppage of tumor growth in living mice through PAI-guided photothermal therapy. This study thus provides an insight into the development of advanced NIR-absorbing small molecules for practical phototheranostic applications.

Intracellular Cascade FRET for Temperature Imaging of Living Cells with Polymeric Ratiometric Fluorescent Thermometers

Intracellular temperature plays a prominent role in cellular functions and biochemical activities inside living cells, but effective intracellular temperature sensing and imaging is still in its infancy. Herein, thermoresponsive double hydrophilic block copolymers (DHBCs)-based fluorescent thermometers were fabricated to investigate their application in intracellular temperature imaging. Blue-emitting coumarin monomer, CMA, green-emitting 7-nitro-2,1,3-benzoxadiazole (NBD) monomer, NBDAE, and red-emitting rhodamine B monomer, RhBEA, were copolymerized separately with N-isopropylacrylamide (NIPAM) to afford dye-labeled PEG-b-P(NIPAM-co-CMA), PEG-b-P(NIPAM-co-NBDAE), and PEG-b-P(NIPAM-co-RhBEA). Because of the favorable fluorescence resonance energy transfer (FRET) potentials between CMA and NBDAE, NBDAE and RhBEA, as well as the slight tendency between CMA and RhBEA fluorophore pairs, three polymeric thermometers based on traditional one-step FRET were fabricated by facile mixing two of these three fluorescent DHBCs, whereas exhibiting limited advantages. Thus, two-step cascade FRET among three polymeric fluorophores was further interrogated, in which NBD acted as a bridging dye by transferring energy from CMA to RhBEA. Through the delicate optimization of the molar contents of three polymeric components, a ∼8.4-fold ratio change occurred in the temperature range of 20-44 °C, and the detection sensitivity improved significantly, reached as low as ∼0.4 °C, which definitely outperformed other one-step FRET thermometers in the intracellular temperature imaging of living cells. To our knowledge, this work represents the first example of polymeric ratiometric thermometer employing thermoresponsive polymer-based cascade FRET mechanism.

Thermoresponsive unimolecular micelles with a hydrophobic dendritic core and a double hydrophilic block copolymer shell

Biocompatible stimuli-responsive unimolecular polymeric micelles have attracted much interest due to their unique structures and potential applications in biomedical fields such as drug delivery and tissue engineering. Here, we report the preparation of dendritic unimolecular polymeric micelles with temperature sensitive shells via reversible addition-fragmentation transfer (RAFT) technique. A multi-arm star amphiphilic copolymer (H40-PDEA) with a hydrophobic hyperbranched polyester (Boltorn H40) as the core and the grafted poly(N,N-diethylacrylamide) (PDEA) as the shell was prepared using H40 based macroRAFT agent. And a dendritic unimolecular polymer (H40-PDEA-PDMA) with a double hydrophilic block copolymer (DHBC) [PDEA-b-poly(2-(dimethylamino)ethyl methacrylate) (PDEA-b-PDMA)] as the dual thermoresponsive shells was synthesized by H40-PDEA based macroRAFT agent. Both H40-PDEA and H40-PDEA-PDMA have a reversible phase transition behavior in aqueous solution. In particular, the unimolecular polymeric micelles H40-PDEA-PDMA with double thermoresponsive shells exhibit a two-stage phase transition behavior. Laser light scattering (LLS), UV-vis transmittance, excimer fluorescence measurements, and micro-differential scanning calorimetry (micro-DSC) were used in combination to probe the conformational changes of chains located at the inner layer and outer corona during the phase transition process.