Extracorporeal Delivery of a Therapeutic Enzyme

To remove circulating harmful small biochemical(s)/substrates causing/deteriorating certain chronic disease, therapeutic enzyme(s) delivered via vein injection/infusion suffer(s) from immunoresponse after repeated administration at proper intervals for a long time and short half-lives since delivery. Accordingly, a novel, generally-applicable extracorporeal delivery of a therapeutic enzyme is proposed, by refitting a conventional hemodialysis device bearing a dialyzer, two pumps and connecting tubes, to build a routine extracorporeal blood circuit but a minimal dialysate circuit closed to circulate the therapeutic enzyme in dialysate. A special quantitative index was derived to reflect pharmacological action and thus pharmacodynamics of the delivered enzyme. With hyperuricemic blood in vitro and hyperuricemic geese, a native uricase via extracorporeal delivery was active in the dialysate for periods much longer than that in vivo through vein injection, and exhibited the expected pharmacodynamics to remove uric acid in hyperuricemic blood in vitro and multiple forms of uric acid in hyperuricemic geese. Therefore, the extracorporeal delivery approach of therapeutic enzymes was effective to remove unwanted circulating small biochemical(s)/substrates, and was expected to avoid immunogenicity problems of therapeutic enzymes after repeated administration at proper intervals for a long time due to no contacts with macromolecules and cells in the body

Continuous and Noninvasive Measurement of Arterial Pulse Pressure and Pressure Waveform using an Image-free Ultrasound System

The local beat-to-beat local pulse pressure (PP) and blood pressure waveform of arteries, especially central arteries, are important indicators of the course of cardiovascular diseases (CVDs). Nevertheless, noninvasive measurement of them remains a challenge in the clinic. This work presents a three-element image-free ultrasound system with a low-computational method for real-time measurement of local pulse wave velocity (PWV) and diameter waveforms, enabling real-time and noninvasive continuous PP and blood pressure waveforms measurement without calibration. The developed system has been well-validated in vitro and in vivo. In in vitro cardiovascular phantom experiments, the results demonstrated high accuracy in the measurement of PP (error < 3 mmHg) and blood pressure waveform (root-mean-square-errors (RMSE) < 2 mmHg, correlation coefficient (r) > textgreater 0.99). In subsequent human carotid experiments, the system was compared with an arterial tonometer, which showed excellent PP accuracy (mean absolute error (MAE) = 3.7 +- 3.4 mmHg) and pressure waveform similarity (RMSE = 3.7 +- 1.6 mmHg, r = 0.98 +- 0.01). Furthermore, comparative experiments with the volume clamp device demonstrated the system's ability to accurately trace blood pressure changes (induced by deep breathing) over a period of one minute, with the MAE of DBP, MAP, and SBP within 5 +- 8 mmHg. The present results demonstrate the accuracy and reliability of the developed system for continuous and noninvasive measurement of arterial PP and blood pressure waveform measurements, with potential applications in the diagnosis and prevention of CVDs.Comment: 13 pages, 12 figure

Nanotechnology-based delivery of CRISPR/Cas9 for cancer treatment

CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-associated protein 9) is a potent technology for gene-editing. Owing to its high specificity and efficiency, CRISPR/Cas9 is extensity used for human diseases treatment, especially for cancer, which involves multiple genetic alterations. Different concepts of cancer treatment by CRISPR/Cas9 are established. However, significant challenges remain for its clinical applications. The greatest challenge for CRISPR/Cas9 therapy is how to safely and efficiently deliver it to target sites in vivo. Nanotechnology has greatly contributed to cancer drug delivery. Here, we present the action mechanisms of CRISPR/Cas9, its application in cancer therapy and especially focus on the nanotechnology-based delivery of CRISPR/Cas9 for cancer gene editing and immunotherapy to pave the way for its clinical translation. We detail the difficult barriers for CRISIR/Cas9 delivery in vivo and discuss the relative solutions for encapsulation, target delivery, controlled release, cellular internalization, and endosomal escape.</p

Two-tiered Online Optimization of Region-wide Datacenter Resource Allocation via Deep Reinforcement Learning

This paper addresses the important need for advanced techniques in continuously allocating workloads on shared infrastructures in data centers, a problem arising due to the growing popularity and scale of cloud computing. It particularly emphasizes the scarcity of research ensuring guaranteed capacity in capacity reservations during large-scale failures. To tackle these issues, the paper presents scalable solutions for resource management. It builds on the prior establishment of capacity reservation in cluster management systems and the two-level resource allocation problem addressed by the Resource Allowance System (RAS). Recognizing the limitations of Mixed Integer Linear Programming (MILP) for server assignment in a dynamic environment, this paper proposes the use of Deep Reinforcement Learning (DRL), which has been successful in achieving long-term optimal results for time-varying systems. A novel two-level design that utilizes a DRL-based algorithm is introduced to solve optimal server-to-reservation assignment, taking into account of fault tolerance, server movement minimization, and network affinity requirements due to the impracticality of directly applying DRL algorithms to large-scale instances with millions of decision variables. The paper explores the interconnection of these levels and the benefits of such an approach for achieving long-term optimal results in the context of large-scale cloud systems. We further show in the experiment section that our two-level DRL approach outperforms the MIP solver and heuristic approaches and exhibits significantly reduced computation time compared to the MIP solver. Specifically, our two-level DRL approach performs 15% better than the MIP solver on minimizing the overall cost. Also, it uses only 26 seconds to execute 30 rounds of decision making, while the MIP solver needs nearly an hour

Improving the knock-in efficiency of the MOF-encapsulated CRISPR/Cas9 system through controllable embedding structures

Appropriate tuning of robust artificial coatings can not only enhance intracellular delivery but also preserve the biological functions of genetic molecules in gene based therapies. Here, we report a strategy to synthesize controllable nanostructures in situ by encapsulating CRISPR/Cas9 plasmids into metal-organic frameworks (MOFs) via biomimetic mineralization. The structure-functionality relationship studies indicate that MOF-coated nanostructures dramatically impact the biological features of the contained plasmids through different embedding structures. The plasmids are homogeneously distributed within the heterogeneous nanoarchitecture and protected from enzymatic degradation. In addition, the plasmid-MOF structure exhibits excellent loading capability, pH-responsive release, and affinity for plasmid binding. Through in vitro assays it was found that the superior MOF vector can greatly enhance cellular endocytosis and endo/lysosomal escape of sheltered plasmids, resulting in successful knock-in of GFP-tagged paxillin genomic sequences in cancer cell lines with high transfection potency compared to our previous studies. Thus, the development of new cost-effective approaches for MOF-based intracellular delivery systems offers an attractive option for overcoming the physiological barriers to CRISPR/Cas9 delivery, which shows great potential for investigating paxillin-associated focal adhesions and signal regulation

Mineralization of pH-Sensitive Doxorubicin Prodrug in ZIF-8 to Enable Targeted Delivery to Solid Tumors

The zeolitic imidazolate framework (ZIF-8), composed of zinc ion and dimethylimidazole, is widely used in drug delivery because of the easy fabrication process and the good biosafety. However, ZIF-8 suffers from low affinity to nonelectric-rich drugs and does not have surface functional groups. Here, to deliver doxorubicin (DOX) with ZIF-8 to specific target sites, DOX was first modified with a pH-sensitive linker containing two carboxyl groups to form the inactive prodrug CAD and subsequently seeded inside ZIF-8 by a 5 min mineralization process. CAD has high affinity to ZIF-8 because of the carboxyl groups and can anchor to the ZIF-8 surface to enable the surface modification with folic acid for tumor targeting. Moreover, the DOX release is precisely controlled by three steps of acidic pH response, with the dissociation of the FA layer, the breakdown of the ZIF-8 structure, and the cleavage of the pH-sensitive linker in prodrug. This novel "prodrug-ZIF-8" strategy has opened a new horizon in drug delivery

Delivery of Protein Kinase A by CRISPRMAX and Its Effects on Breast Cancer Stem-Like Properties

Protein kinase A (PKA) activation has recently been reported to inhibit epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) ability, which is considered to be responsible for chemoresistance and tumor recurrence in patients. While current studies mainly focus on gene manipulation of the EMT process, the direct delivery of PKA enzymes to cancer cells has never been investigated. Here, we utilize the commercial Lipofectamine CRISPRMAX reagent to directly deliver PKAs to breast cancer cells and evaluate its effects on EMT regulation. We optimized the delivery parameters with fluorescent-labeled bovine serum albumin, and successfully delivered fluorescent PKAs through CRISPRMAX into breast cancer cells. Then, we evaluated the biological effects by immunofluorescence, flow cytometry, mammosphere assay, and chemoresistance assay. Our data showed the expression of EMT-related markers, alpha-smooth muscle actin and N-cadherin, was downregulated after CRISPRMAX-PKA treatment. Although the CD44(+)/CD24(-) population did not change considerably, the size of mammospheres significantly decreased. In paclitaxel and doxorubicin chemoresistance assays, we noticed PKA delivery significantly inhibited paclitaxel resistance rather than doxorubicin resistance. Taken together, these results suggest our direct enzyme delivery can be a potential strategy for inhibiting EMT/CSC-associated traits, providing a safer approach and having more clinical translational efficacy than gene manipulation. This strategy will also facilitate the direct testing of other target enzymes/proteins on their biological functions

Microfluidic-assisted biomineralization of CRISPR/Cas9 in near-infrared responsive metal-organic frameworks for programmable gene-editing

Ribonucleoprotein (RNP) based CRISPR/Cas9 gene-editing system shows great potential in biomedical applications. However, due to the large size, charged surface and high biological sensitivity of RNP, its efficient delivery with precise control remains highly challenging. Herein, a microfluidic-assisted metal-organic framework (MOF) based biomineralization strategy is designed and utilized for the efficient delivery and remote regulation of CRISPR/Cas9 RNP gene editing. The strategy is realized by biomimetic growing of thermo-responsive EuMOFs onto photothermal template Prussian blue (PB). The RNP is loaded during MOFs crystallization in microfluidic channels. By adjusting different microfluidic parameters, well-defined and comparable RNP encapsulated nanocarrier (PB@RNP-EuMOFs) are obtained with high loading efficiency (60%), remarkable RNP protection and NIR-stimulated release capacity. Upon laser exposure, the nanocarrier induces effective endosomal escape (4 h) and precise gene knockout of green fluorescent protein by 40% over 2 days. Moreover, the gene-editing activity can be programmed by tuning exposure times (42% for three times and 47% for four times), proving more controllable and inducible editing modality compared to control group without laser irradiation. This novel microfluidic-assisted MOFs biomineralization strategy thus offers an attractive route to optimize delivery systems and reduce off-target side effects by NIR-triggered remote control of CRISPR/Cas9 RNP, improving the potential for its highly efficient and precise therapeutic application

Effective Delivery of the CRISPR/Cas9 System Enabled by Functionalized Mesoporous Silica Nanoparticles for GFP-Tagged Paxillin Knock-In

In this study, direct and effective intracellular delivery of CRISPR/Cas9 plasmids for homology-directed repair is achieved by functionalized mesoporous silica nanoparticles (MSNs). The functionalized MSNs (Cy5.5-MSNs-NLS) are synthesized by in situ labeling of a fluorescent dye (Cy5.5) and surface conjugation of nuclear localization sequence (NLS, PKKKRKV), showing a high loading efficiency (50%) toward the plasmids (PXN cutdown plasmid: GFP-Cas9-paxillin_gRNA and repair plasmid: AICSDP-1: PXN-EGFP). Subsequently, a polymeric coating of the poly(dimethyldiallylammonium chloride) (PDDA) is electrostatically deposited onto the plasmid-loaded Cy5.5-MSNs-NLS by microfluidic nanoprecipitation. The coating layer offers effective protection against the denaturation of plasmids by EcoRV restriction enzymes, and is shown to prevent premature release. Moreover, owing to the positive charge and pH-responsive disaggregation of PDDA, enhanced cellular internalization (16 h) and endosomal escape (4 h) of the nanocarrier are observed. After escape of nanocarrier system into the cytoplasm, the NLS on the surface of MSNs facilitates nuclear transport of the CRISPR/Cas9 plasmids, achieving successful GFP-tag knock-in of the PXN genomic sequence in U2OS cells. This intracellular delivery system thus offers an attractive method to overcome physiological barriers for CRISPR/Cas9 delivery, showing considerable promise for paxillin-associated focal adhesion and signaling regulator investigation