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Stimuli-responsive biomaterials that contain logic gates hold great potential for detecting and responding to pathological markers as part of clinical therapies. However, a major barrier is the lack of a generalized system that can be used to easily assemble different ligand-responsive units to form programmable nanodevices for advanced biocomputation. Here we develop a programmable polymer library by including responsive units in building blocks with similar structure and reactivity. Using these polymers, we have developed a series of smart nanocarriers with hierarchical structures containing logic gates linked to self-immolative motifs. Designed with disease biomarkers as inputs, our logic devices showed site-specific release of multiple therapeutics (including kinase inhibitors, drugs and short interfering RNA) in vitro and in vivo. We expect that this ‘plug and play’ platform will be expanded towards smart biomaterial engineering for therapeutic delivery, precision medicine, tissue engineering and stem cell therapy.A programmable polymer library that responds to external and internal stimuli has been developed and used to fabricate a series of nanocarriers for drug release. The carriers respond to disease biomarkers, triggering self-immolative motifs and leading to the site-specific release of therapeutics both in vitro and in vivo.

Proteolysis targeting chimeras (PROTACs) represent an emerging targeted cancer therapy approach. However, their poor cell penetration and instability in vivo pose daunting challenges for wide‐spread clinical usage. To enhance the in vivo therapeutic efficacy of PROTACs, we introduced extracellular vesicles (EVs) for in vivo PROTAC delivery, which is leveraged by a novel microfluidic droplet‐based EV electro‐transfection system (μDES). We previously developed YX968 PROTAC, which can selectively degrade both HDAC3 and HDAC8 in triple negative breast cancer (TNBC) cells and effectively suppress the tumour cell growth without provoking global hyperacetylation. In this manuscript, we demonstrated that YX968 loaded EVs via the μDES system can retain the optimal integrity of drug loaded EVs with improved loading efficiency compared to other transfection approaches, which, in turn, significantly enhances the therapeutic function of PROTAC in vivo in TNBC mouse models. Intraperitoneal injections of YX968 loaded EVs led to significantly enhanced intratumoral degradation of HDAC3 and HDAC8 than YX986 alone, which resulted in advanced TNBC tumour inhibition without noticeable tissue toxicity. Such EV‐based delivery strategy, with a scalable EV loading approach, enhanced the in vivo PROTAC drug stability and bioavailability and improved tissue penetration and targeting, filling an important gap in the clinical translation of PROTAC‐based cancer therapy. We introduce an extracellular vesicles (EVs) based in vivo PROTAC delivery platform, which is leveraged by a novel microfluidic droplet EV electro‐transfection system (μDES), enabling large scale production. The in vivo therapeutic efficacy for treating triple negative breast cancer is significantly enhanced with improved safety, which fills an important gap in the clinical translation of PROTAC‐based cancer therapy.

Adding synthetic nucleotides to DNA increases the linear information density of DNA molecules. Here we report that it also can increase the diversity of their three-dimensional folds. Specifically, an additional nucleotide (dZ, with a 5-nitro-6-aminopyridone nucleobase), placed at twelve sites in a 23-nucleotides-long DNA strand, creates a fairly stable unimolecular structure (that is, the folded Z-motif, or fZ-motif) that melts at 66.5 °C at pH 8.5. Spectroscopic, gel and two-dimensional NMR analyses show that the folded Z-motif is held together by six reverse skinny dZ − :dZ base pairs, analogous to the crystal structure of the free heterocycle. Fluorescence tagging shows that the dZ − :dZ pairs join parallel strands in a four-stranded compact down–up–down–up fold. These have two possible structures: one with intercalated dZ − :dZ base pairs, the second without intercalation. The intercalated structure would resemble the i-motif formed by dC:dC + -reversed pairing at pH ≤ 6.5. This fZ-motif may therefore help DNA form compact structures needed for binding and catalysis. Standard DNA is limited by low information density and functional diversity. Now it has been shown that an expanded genetic alphabet—incorporating a synthetic nucleotide, dZ—allows for the creation of stable three-dimensional DNA structures under mild alkaline conditions. Such stable structures enrich our understanding of DNA’s structural diversity and its potential in synthetic biology applications.

Nucleic acid aptamers, also known as “chemical antibodies”, generated from cell-based Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX), can be used to recognize specific biomarkers on the cell membrane with high affinity and specificity. Nucleic acid aptamers can be coupled to proteins in order to target those proteins to specific cell types. In comparison to other targeting strategies, aptamers have many advantages such as a small size and low immunogenicity, and they can be easily modified chemically.This dissertation investigated whether conjugating DNA aptamer to a variety of functional proteins could be used to significantly enhanced the targeted delivery of functional proteins in cancer therapy. Firstly, a bispecific circular aptamer (bc-apt) composed of a cell-specific aptamer and an anti-His tag aptamer, showed good binding properties to specific cells as well as to the polyhistidine tag on functional proteins. By noncovalently and stably tethering His-proteins with specific biomarkers on cancer cells, bc-apt was demonstrated to enhance the intracellular delivery of His-EGFP for the cell imaging and His-RNase A for a better therapeutic outcome. However, it was also found that the binding performance of bc-apt was affected by protein size and the surface charge of the proteins. To tackle the unmet need of targeted delivery of the larger proteins such as the gene editing protein Cas9, aptamer-Cas9 bioconjugates were therefore constructed using the amine-reactive homo-bifunctional crosslinker disuccinimidyl suberate (DSS) and then demonstrated to specifically and efficiently bind with and enter the targeted cells. DSS crosslinking reagents were shown to efficiently conjugate Cas9 with DNA aptamers with different DNA to protein ratios, however, the resulting aptamer-Cas9 bioconjugates showed a low level of undesired bioactivity in both cell-free and in vitro conditions compared to wild-type Cas9. In order to prevent the adverse effects of bioconjugation, we proposed that the bifunctional cleavable linker AzMMMan, which was reported to be hydrolyzed in a mild acidic environment like endosomes, is adequate to construct reversible aptamer-Cas9 bioconjugates for the desired targeted gene editing in cancer therapy.Consequently, all results in the dissertation showed that therapeutic proteins were efficiently conjugated with DNA aptamers via chemical crosslinkers or aptamer-His tag interaction, and the resulting DNA-protein complexes in both strategies were promising in enhancing the targeted delivery of various functional proteins for cancer therapy.

: A cascade, or domino, reaction consists of two, or more, consecutive reactions such that subsequent reactions occur only if some chemical functionality has first been established in the prior step. However, while construction of predesigned and desired molecular domino reactors in a tailored manner is a valuable endeavor, it is still challenging. : To address this challenge, we herein report an aptamer-based photodynamic domino reactor built through automated modular synthesis. The engineering of this reactor takes advantage of the well-established solid-phase synthesis platform to incorporate a photosensitizer into G-quadruplex/ hemin DNAzyme at the molecular level. : As a proof of concept, our photodynamic domino reactor, termed AS1411/hemin- pyrochlorophyll A, achieves photodynamic domino reaction for efficient cancer treatment by using a high concentration of hydrogen peroxide (H O ) in the tumor microenvironment (TME) to produce O , followed by consecutive generation of singlet oxygen ( O ) using the pre-produced O . More specifically, phosphoramidite PA (pyrochlorophyll A) is coupled to aptamer AS1411 to form AS1411-PA ApDC able to simultaneously perform targeted imaging and photodynamic therapy (PDT). The insertion of hemin into the AS1411 G-quadruplex was demonstrated to alleviate tumor hypoxia by decomposition of H O to produce O . This was followed by the generation of O by PA to trigger cascading amplified PDT. : Therefore, this study provides a general strategy for building an aptamer-based molecular domino reactor through automated modular synthesis. By proof of concept, we further demonstrate a novel method of achieving enhanced PDT, as well as alleviating TME hypoxia at the molecular level.

Background and objective While the six-minute walk test (6MWT) is often used to assess exercise capacity, the less well-known two-minute walk test (2MWT) is more feasible for some patients. In previous studies, we developed reference equations for the two-minute walk distance (2MWD) for healthy Chinese adults. However, our study did not recruit people with obesity, and the reference equations did not apply to participants with a body mass index (BMI) > 30 kg/m2. The main objective of this study was to establish reference equations for the 2MWD among middle-aged and elderly Chinese individuals with obesity. Methods A total of 295 individuals were recruited. The participants underwent two 2MWTs, with the longer of the two 2MWDs used for further analyses. The reference equations for the 2MWD were developed using stepwise multiple regression analysis. The newly established equations for the 2MWD were then compared with the existing equations. Results The mean 2MWD of the participants was 176±20 m. Age and BMI were identified as independent factors that influenced the 2MWD and explained 28% and 32% of the variance in walking distance for the male and female groups, respectively. The reference equations for the 2MWD were as follows: Conclusion This study resulted in the development of reference equations for predicting 2MWD among middle-aged and elderly Chinese people with obesity. These equations will be a clinically valuable tool for evaluating functional capacity, determining prognoses and monitoring treatment in middle-aged and elderly Chinese people with obesity.

Familial mutations in C99 can increase the total level of the soluble Aβ peptides produced by proteolysis, as well as the Aβ42/Aβ40 ratio, both of which are linked to the progression of Alzheimer’s disease. We show that the extracellular sequence of C99 forms β-sheet structure upon interaction with membrane bilayers. Mutations that disrupt this structure result in a significant increase in Aβ production and, in specific cases, result in an increase in the amount of Aβ42 relative to Aβ40. Fourier transform infrared and solid-state NMR spectroscopic studies reveal a central β-hairpin within the extracellular sequence comprising Y10-E11-V12 and L17-V18-F19 connected by a loop involving H13-H14-Q15. These results suggest how familial mutations in the extracellular sequence influence C99 processing and provide a structural basis for the development of small molecule modulators that would reduce Aβ production.

Proteolysis targeting chimeras (PROTACs) are an emerging targeted cancer therapy approach, but wide-spread clinical use of PROTAC is limited due to poor cell targeting and penetration, and instability in vivo. To overcome such issues and enhance the in vivo efficacy of PROTAC drugs, microfluidic droplet-based electroporation (µDES) was developed as a novel extracellular vesicle (EVs) transfection system, which enables the high-efficient PROTAC loading and effective delivery in vivo. Our previously developed YX968 PROTAC drug had shown the selectively degradation of HDAC3 and 8, which effectively suppresses the growth of breast tumor cell lines, including MDA-MB-231 triple negative breast cancer (TNBC) line, via dual degradation without provoking a global histone hyperacetylation. In this study, we demonstrated that µDES-based PROTAC loading in EVs significantly enhanced therapeutic function of PROTAC drug in vivo in the TNBC breast tumor mouse model. NSG mice with pre-established MDA-MB-231 tumors and treated with intraperitoneal injection of EVs for tumor inhibition study, which showed significantly higher HDAC 3 and 8 degradation efficiency and tumor inhibition than PROTAC only group. The liver, spleen, kidney, lung, heart, and brain were collected for safety testing, which exhibited improved toxicity. The EV delivery of PROTAC drug enhances drug stability and bioavailability in vivo, transportability, and drug targeting ability, which fills an important gap in current development of PROTAC therapeutic functionality in vivo and clinical translation. This novel EV-based drug transfection and delivery strategy could be applicable to various therapeutics for enhancing in vivo delivery, efficacy, and safety.

Clinical translation of gene therapy has been challenging, due to limitations in current delivery vehicles such as traditional viral vectors. Herein, we report the use of gRNA:Cas9 ribonucleoprotein (RNP) complexes engineered extracellular vesicles (EVs) for in vivo gene therapy. By leveraging a novel high-throughput microfluidic droplet-based electroporation system (uDES), we achieved 10-fold enhancement of loading efficiency and more than 1000-fold increase in processing throughput on loading RNP complexes into EVs (RNP-EVs), compared with conventional bulk electroporation. The flow-through droplets serve as enormous bioreactors for offering millisecond pulsed, low-voltage electroporation in a continuous-flow and scalable manner, which minimizes the Joule heating influence and surface alteration to retain natural EV stability and integrity. In the Shaker-1 mouse model of dominant progressive hearing loss, we demonstrated the effective delivery of RNP-EVs into inner ear hair cells, with a clear reduction of Myo7ash1 mRNA expression compared to RNP-loaded lipid-like nanoparticles (RNP-LNPs), leading to significant hearing recovery measured by auditory brainstem responses (ABR).Competing Interest StatementThe authors have declared no competing interest.Footnotes* More animal work was done to solidify the conclusions and updated in this version in Figure 4 and Figure 5.