Construction of Tough Hydrogel Cross-Linked via Ionic Interaction by Protection Effect of Hydrophobic Domains.

Most hydrogels have poor mechanical properties, severely limiting their potential applications, and numerous approaches have been introduced to fabricate more robust and durable examples. However, these systems consist of nonbiodegradable polymers which limit their application in tissue engineering. Herein, we focus on the fabrication and investigate the influence of hydrophobic segments on ionic cross-linking properties for the construction of a tough, biodegradable hydrogel. A biodegradable, poly(γ-glutamic acid) polymer conjugated with a hydrophobic amino acid, l-phenylalanine ethyl ester (Phe), together with an ionic cross-linking group, alendronic acid (Aln) resulting in γ-PGA-Aln-Phe, was initially synthesized. Rheological assessments through time sweep oscillation testing revealed that the presence of hydrophobic domains accelerated gelation. Comparing gels with and without hydrophobic domains, the compressive strength of γ-PGA-Aln-Phe was found to be six times higher and exhibited longer stability properties in ethylenediaminetetraacetic acid solution, lasting for up to a month. Significantly, the contribution of the hydrophobic domains to the mechanical strength and stability of ionic cross-linking properties of the gel was found to be the dominant factor for the fabrication of a tough hydrogel. As a result, this study provides a new strategy for mechanical enhancement and preserves ionic cross-linked sites by the addition of hydrophobic domains. The development of tough, biodegradable hydrogels reported herein will open up new possibilities for applications in the field of biomaterials.

Engineering metabolic cycle-inspired hydrogels with enzyme-fueled programmable transient volume changes.

An enzyme-fueled transient volume phase transition (TVPT) of hydrogels under out-of-equilibrium conditions is reported. The approach takes inspiration from the metabolic cycle, comprising nutrient intake and anabolism/catabolism followed by waste excretion. The incorporation of methacrylic acid and acrylated trypsin in a polymeric hydrogel allowed the TVPT of the gel to be fueled by lysozyme. With the intake of lysozyme as fuel, the construction/destruction of electrostatic cross-linkages induced transient shrinkage/swelling of the gel accompanied by the depletion of lysozyme activity. The system's transient response could be flexibly programmed by adjusting not only the fuel concentration but the chemical composition of materials. The lysozyme-fueled TVPT of the gel could be exploited to transient changes in the mechanical properties of the gel. Our work opens a route toward a new class of stimuli-responsive hydrogels for biomedical applications.

Fabrication of Molecular Blocks with High Responsiveness to the Cancer Microenvironment by Ursodeoxycholic Acid.

In cancer therapy, a drug delivery system (DDS) has been widely studied to achieve selective drug accumulation at the tumor site. However, DDS still has a major drawback in that it requires multistep processes for intracellular delivery, resulting in low efficiency of drug delivery. To overcome this problem, we recently reported a molecular block (MB) that disrupts cancer cell membranes in the cancer microenvironment using deoxycholic acid (DCA). However, the MB showed considerable cytotoxicity even at neutral pH, possibly due to the structural hydrophobic property of DCA. Herein, we focused on selecting the most suitable bile acid for an MB that possessed high responsiveness to the cancer microenvironment without cytotoxicity at neutral pH. Cell viabilities of the free bile acids such as DCA, chenodeoxycholic acid (CDCA), cholic acid (CA), and ursodeoxycholic acid (UDCA) were evaluated at neutral pH (pH = 7.4) and a cancer acidic environment (pH = 6.3-6.5). The half-maximal inhibition concentration (IC50) value of UDCA at pH = 7.4 showed an approximately 7.5-fold higher IC50 value than that at pH = 6.3, whereas the other bile acids yielded less than a 4-fold IC50 value difference between the same pHs. Biocompatible poly(vinyl alcohol) (PVA) was functionalized with UDCA (PVA-UDCA) for the synthesis of higher responsiveness to the cancer microenvironment without cytotoxicity at neutral pH. Importantly, 56% pancreatic cancer cell death was observed at pH = 6.5, whereas only 10% was detected at neutral pH by the PVA-UDCA treatment. However, PVA-DCA indicated almost the same cancer cell death property, independent of pH conditions. These results suggest PVA-UDCA shows great potential for a new class of MB.

Fabrication of a Polymeric Inhibitor of Proximal Metabolic Enzymes in Hypoxia for Synergistic Inhibition of Cancer Cell Proliferation, Survival, and Migration.

Since conventional molecular targeted drugs often result in side effects, the development of novel molecular targeted drugs with both high efficacy and selectivity is desired. Simultaneous inhibition of metabolically and spatiotemporally related proteins/enzymes is a promising strategy for improving therapeutic interventions in cancer treatment. Herein, we report a poly-α-l-glutamate-based polymer inhibitor that simultaneously targets proximal transmembrane enzymes under hypoxia, namely, carbonic anhydrase IX (CAIX) and zinc-dependent metalloproteinases. A polymer incorporating two types of inhibitors more effectively inhibited the proliferation and migration of human breast cancer cells than a combination of two polymers functionalized exclusively with either inhibitor. Synergistic inhibition of cancer cells would occur owing to the hetero-multivalent interactions of the polymer with proximate enzymes on the cancer cell membrane. Our results highlight the potential of polymer-based cancer therapeutics.

Biomacromolecule-Fueled Transient Volume Phase Transition of a Hydrogel.

A metabolic cycle-inspired hydrogel which exhibits the biomacromolecule-fueled transient volume phase transition is reported. This hydrogel has the affinity and digestive capacity for a fuel α-poly-L-lysine by incorporating acrylic acid and trypsin. The hydrogel captured fuel and transiently shrank owing to the construction of electrostatic cross-linkages. This process was inherently connected with the digestion of these cross-linkages and the release of oligo-lysine as waste, which induced the reswelling of the hydrogel at equilibrium. The transient volume change of the hydrogel realized the fuel-stimulated transient release of a payload. This study provides a strategy for engineering materials with biomacromolecule-fueled dynamic functions under the out-of-equilibrium condition.

Fabrication of highly stretchable hydrogel based on crosslinking between alendronates functionalized poly-γ-glutamate and calcium cations.

We report a highly stretchable hydrogel based on the crosslinking structure between calcium cations and alendronates (ALN) conjugated with poly-γ-glutamate (γ-PGA), a typical biodegradable polymer. γ-PGA with ALN (γ-PGA-ALN) forms the hydrogel in the aqueous solution containing CaCl2. The hydrogel shows 2000% of stretchability and reversible stretching without failure at a strain of 250%. The fracture strain and stress are tunable by varying the concentration of either γ-PGA-ALN or CaCl2, indicating the importance of fine-tuning of the density of the cross-linkage to control the mechanical properties of the hydrogel. We believe the biodegradable polymer based highly stretchable hydrogel has potential for use in various fields such as tissue engineering.

Development of Highly Sensitive Molecular Blocks at Cancer Microenvironment for Rapid Cancer Cell Death.

Improving the efficiency and selectivity of drug delivery systems (DDS) is still a major challenge in cancer therapy. Recently, the low transport efficiency of anticancer drugs using a nanocarrier due to the elimination of the carriers from the blood circulation and the blocking by tumor stromal tissues surrounding cancer cells has been reported. Furthermore, multiple steps are required for their intracellular delivery. We recently reported a cancer microenvironment-targeting therapy termed molecular block (MB) which induced cancer cell death by a pH-driven self-aggregation and cell membrane disruption at tumor microenvironment. The MB were designed to disperse as nanoscale assemblies in the bloodstream for efficient circulation and penetration through the stromal tissues. When the MBs reach the tumor site, they self-assembled in microscale aggregates on the cancer cell surfaces in response to the cancer microenvironment and induced cancer cell death. However, in vivo study in mice showed that the MB could not efficiently accumulate at the tumor site because slight hydrophobic aggregations in the bloodstream might potentially be the reason for the off-target accumulation. In this study, we optimize the hydrophilic-hydrophobic balance of MB for avoiding the off-target accumulation and for gaining higher sensitivity to the cancer microenvironment at weak acid condition. Copper-free click reaction with propiolic acid was used to reduce the hydrophobicity of the main chain and obtain higher responsive MB at cancer microenvironment for rapid cell killing. The optimized MB can be considered as a promising approach for an improved cancer cell targeting.

A Biomimetic of Endogenous Tissue Inhibitors of Metalloproteinases: Inhibition Mechanism and Contribution of Composition, Polymer Size, and Shape to the Inhibitory Effect.

A biomimetic of endogenous tissue inhibitors of metalloproteinases (TIMPs) was engineered by introducing three binding elements to a synthetic tetrapolymer. We evaluated the contribution of composition, size, and shape of the TIMP-mimicking polymers to the inhibition of BaP1, a P-I class snake venom metalloproteinase (SVMP). Inhibition was achieved when the size of the linear polymer (LP) was comparable to or greater than that of the enzyme, indicating the efficacy requires binding to a significant portion of the enzyme surface in the vicinity of the active site. The efficacy of a low cross-linked polymer hydrogel nanoparticle (NP) of substantially greater molecular weight was comparable to that of the LPs despite differences in size and shape, an important finding for in vivo applications. The abiotic TIMP was effective against two classes of SVMPs in whole snake venom. The results can serve as a design principle for biomimetic polymer inhibitors of enzymes.

Affinity purification of multifunctional oligomeric ligands synthesized via controlled radical polymerization.

Abiotic oligomeric ligands with a strong affinity for a target peptide sequence were isolated by affinity purification from a pool of 30-mer acrylic random ter-oligomers that were synthesized via a controlled radical polymerization process. Our results indicate that the oligomeric ligands with suitable sequence and/or stereochemical configurations for the target can be isolated from as-polymerized random co-polymers based on the affinity to the target. This process will be a powerful tool for the development of stable and inexpensive ligands that can be used to detect, neutralize and purify proteins with a target epitope sequence.

Abiotic Mimic of Endogenous Tissue Inhibitors of Metalloproteinases: Engineering Synthetic Polymer Nanoparticles for Use as a Broad-Spectrum Metalloproteinase Inhibitor.

We describe a process for engineering a synthetic polymer nanoparticle (NP) that functions as an effective, broad-spectrum metalloproteinase inhibitor. Inhibition is achieved by incorporating three functional elements in the NP: a group that interacts with the catalytic zinc ion, functionality that enhances affinity to the substrate-binding pocket, and fine-tuning of the chemical composition of the polymer to strengthen NP affinity for the enzyme surface. The approach is validated by synthesis of a NP that sequesters and inhibits the proteolytic activity of snake venom metalloproteinases from five clinically relevant species of snakes. The mechanism of action of the NP mimics that of endogenous tissue inhibitors of metalloproteinases. The strategy provides a general design principle for synthesizing abiotic polymer inhibitors of enzymes.

On-site evaluation of CT-based fractional flow reserve using simple boundary conditions for computational fluid dynamics.

Fractional flow reserve (FFR) is an established method for diagnosing physiological coronary artery stenosis. A method for computing FFR using coronary computed tomography (CT) images was recently developed. However, its calculation requires off-site supercomputer analysis. Here, we report the preliminary result of a method using simple estimation of boundary conditions. The lumen boundaries of the coronary arteries were semi-automatically delineated using full width at half maximum of CT number profiles. The computational fluid dynamics (CFD) of the blood flow was performed using the boundary conditions of a fixed pressure at the coronary ostium and flow rates at each outlet. The total inflow at the coronary ostium was estimated based on the uniform wall shear stress hypothesis and corrected using a hyperemic multiplier to gain a hyperemic flow rate. The flow distribution from a parent vessel to the downstream daughter vessels was determined according to Murray's law. FFR estimated by CFD was calculated as FFRCFD = Pd/Pa. We collected patients who underwent coronary CT and coronary angiography followed by invasively measured FFR and compared FFRCFD with FFR. Sensitivity, specificity, and correlations were assessed. A total of 48 patients and 72 arteries were assessed. The correlation coefficient of FFRCFD with FFR was 0.56. The cut-off value was ≤ 0.80, sensitivity was 59.1%, and specificity was 94.0%. CFD-based FFR using simple boundary conditions for on-site clinical computation provided FFRCFD values that were moderately correlated with invasively measured FFR.

Engineering the Binding Kinetics of Synthetic Polymer Nanoparticles for siRNA Delivery.

The affinity of a synthetic polymer nanoparticle (NP) to a target biomacromolecule is determined by the association and dissociation rate constants (kon, koff) of the interaction. The individual rates and their sensitivity to local environmental influences are important factors for the on-demand capture and release a target biomacromolecule. Positively charged NPs for small interfering RNA (siRNA) delivery is a case in point. The knockdown efficacy of siRNA can be strongly influenced by the binding kinetics to the NP. Here, we show that kon and koff of siRNA to NPs can be individually engineered by tuning the chemical structure and composition of the NP. N-Isopropylacrylamide-based NPs functionalized with hydrophobic and amine monomers were used. koff decreased by increasing the amount of amine groups in the NP, whereas kon did not change. Importantly, NPs showing a low koff at pH 5.5 together with a high koff at pH 7.4 showed high knockdown efficiency when NP/siRNA complexes were packaged in lipid nanoparticles. These results provide direct evidence for the premise that the efficacy of an siRNA delivery vector is linked with the strong affinity to the siRNA in the endosome and low affinity in the cytoplasm.

Reversible p Ka Modulation of Carboxylic Acids in Temperature-Responsive Nanoparticles through Imprinted Electrostatic Interactions.

The acid dissociation constants (p Ka values) of Brønsted acids at the active sites of proteins are reversibly modulated by intramolecular electrostatic interactions with neighboring ions in a reaction cycle. The resulting p Ka shift is crucial for the proteins to capture, transfer, and release target ions. On the other hand, reversible p Ka modulation through electrostatic interactions in synthetic polymer materials has seldom been realized because the interactions are strongly shielded by solvation water molecules in aqueous media. Here, we prepared hydrogel nanoparticles (NPs) bearing carboxylic acid groups whose p Ka values can be reversibly modulated by electrostatic interactions with counterions in the particles. We found that the deprotonated states of the acids were stabilized by electrostatic interactions with countercations only when the acids and cations were both imprinted in hydrophobic microdomains in the NPs during polymerization. Cationic monomers, like primary amine- and guanidium group-containing monomers, which interacted strongly with growing NPs showed greater p Ka modulation than monomers that did not interact with the NPs, such as quaternary ammonium group-containing monomers. Modulation was enhanced when the guanidium moieties were protected with hydrophobic groups during polymerization, so that the guanidium ions were imprinted in the hydrophobic microdomains; the lowest p Ka of ∼4.0 was achieved as a result. The p Ka modulation of the acids could be reversibly removed by inducing a temperature-dependent volume phase transition of the gel NPs. These design principles are applicable to other stimuli-responsive materials and integral to the development of synthetic materials that can be used to capture, transport, and separate target ions.

Rational designing of an antidote nanoparticle decorated with abiotic polymer ligands for capturing and neutralizing target toxins.

Many of macromolecular toxins induce cell death by directly interacting with cells or induction of inflammatory cytokines. Abiotic polymer ligands (PLs) composed of functional monomers are able to bind and neutralize toxins in vivo and are of great interest for efficient antidotes. However, little has been reported about recognition and neutralization of target molecules in the bloodstream because of readily elimination from the bloodstream. Here, we report a rational design of PLs-decorated lipid nanoparticles (PL-NPs) for neutralizing a target toxin in vivo. PL that decorated on the NPs would cooperatively interacts with target biomacromolecules since the lipid molecules in NPs have a high degree of freedom. In the present study, N-isopropylacrylamide based PLs interacting with histones, major mediators of sepsis, were synthesized. Affinity between PL-NPs and histones depends on monomer composition and polymer length. The optimized PL-NP showed little affinity for plasma proteins. The PL-NPs inhibited the toxicity of histones both in vitro and in vivo, suggesting that PLs on the NPs cooperatively bound to histones and neutralized their toxicity. In addition, circulation time of optimized PL was significantly prolonged by the modification onto NPs. These results provide a platform for designing antidote nanoparticles neutralizing toxic biomacromolecules.

Wide-range pKa tuning of proton imprinted nanoparticles for reversible protonation of target molecules via thermal stimuli.

pKa tuning of Brønsted acids in synthetic nano-materials is of great importance for the design of ion exchange and bio-/molecular-separation media and polymer catalysis. It has been reported that hydrogel nanoparticles with carboxylic acids that show large and reversible pKa shifts in response to thermal stimuli can be prepared by copolymerization of N-isopropylacrylamide (NIPAm), acrylic acids (AAc) and N,N'-methylene bisacrylamide (BIS) via the proton imprinting polymerization process. However, the reported range of pKa shifts is limited to the range of 5.3 to 7.5. In this study, we report a procedure to prepare proton imprinted NPs that show pKa shifts in the tuned pKa range and demonstrate applications of the NPs. pKa values ranging from 4.3 to 8.7 were achieved by designing the structure of monomers containing carboxylic acids and applying the proton imprinting procedure. It was demonstrated that proton-imprinted NPs with different pKa values could be used for the reversible and selective protonation of target molecules which have specific pKa values. Our results establish the generality of the proton imprinting procedure and provide a guide for designing stable and inexpensive materials for sophisticated purification processes.

CT-based automated planning of acetabular cup for total hip arthroplasty (THA) based on hybrid use of two statistical atlases.

PURPOSE: This study describes the use of CT images in atlas-based automated planning methods for acetabular cup implants in total hip arthroplasty (THA). The objective of this study is to develop an automated cup planning method considering the statistical distribution of the residual thickness. METHODS: From a number of past THA planning datasets, we construct two statistical atlases that represent the surgeon's expertise. The first atlas is a pelvis-cup merged statistical shape model (PC-SSM), which encodes global spatial relationships between the patient anatomy and implant. The other is a statistical residual thickness map (SRTM) of the implant surface, which encodes local spatial constraints of the anatomy and implant. In addition to PC-SSM and SRTM, we utilized the minimum thickness as a threshold constraint to prevent penetration. RESULTS: The proposed method was applied to the pelvis shapes segmented from CT images of 37 datasets of osteoarthritis patients. Automated planning results with manual segmentation were compared to the plans prepared by an experienced surgeon. There was no significant difference in the average cup size error between the two methods (1.1 and 1.2 mm, respectively). The average positional error obtained by the proposed method, which integrates the two atlases, was significantly smaller (3.2 mm) than the previous method, which uses single atlas (3.9 mm). In the proposed method with automated segmentation, the size error of the proposed method for automated segmentation was comparable (1.1 mm) to that for manual segmentation (1.1 mm). The average positional error was significantly worse (4.2 mm) than that using manual segmentation (3.2 mm). If we only consider mildly diseased cases, however, there was no significance between them (3.2 mm in automated and 2.6 mm in manual segmentation). CONCLUSION: We infer that integrating PC-SSM and SRTM is a useful approach for modeling experienced surgeon's preference during cup planning.

Design of Synthetic Polymer Nanoparticles That Facilitate Resolubilization and Refolding of Aggregated Positively Charged Lysozyme.

Designed polymer hydrogel nanoparticles (NPs) capable of facilitating resolubilization and refolding of an aggregated protein, positively charged lysozyme, are prepared. NPs designed to interact strongly with denatured lysozyme and relatively weakly with native lysozyme, facilitated resolubilization and refolding of aggregated lysozyme. Such NPs could be prepared by copolymerizing optimized combinations and populations of functional monomers. The refolded lysozyme showed native conformation and enzymatic activity. Eleven grams of aggregated protein was refolded by 1 g of NPs. However, NPs having low affinity to denatured lysozyme and NPs having high affinity to both denatured and native lysozyme showed relatively low facilitation activity. Our results suggest a potential strategy for the design of artificial chaperones with high facilitating activity.

Morphological analysis of the effects of intraoperative transrectal compression of the prostate during high-intensity focused ultrasound for localized prostate cancer.

OBJECTIVES: To evaluate the effects of transrectal compression of the prostate for intra-operative prostatic swelling and intraprostatic point shift during high-intensity focused ultrasound treatment of localized prostate cancer. METHODS: Patients treated with whole-gland high-intensity focused ultrasound as primary monotherapy for localized prostate cancer were enrolled in the study. Using the standard and compression method, the volumes of degassed water in the balloon covering the high-intensity focused ultrasound probe were 50 mL and 80-160 mL, respectively. To identify prostatic swelling and shift during high-intensity focused ultrasound and the volume occupied by the non-enhanced area, three-dimensional prostate models were reconstructed using ultrasound and contrast-enhanced magnetic resonance imaging. RESULTS: In comparison with the standard (n = 40) and compression (n = 48) methods, intraoperative increase in the prostate volume (21% vs 5.3%; P = 0.044), intraprostatic point shift (4 mm vs 2 mm, P = 0.040 in the transition zone; 3 mm vs 0 mm; P = 0.001 in the peripheral zone) and the volume occupied by the non-enhanced area (89% vs 96%; P = 0.001) were significantly suppressed. The biochemical disease-free survival rate in patients treated using the compression method was significantly improved relative to the standard method (92.6% vs 76.5%; P = 0.038). Regarding complications, there was no significant difference in the rate of urethral stricture (P = 0.9), urinary tract infection (P = 0.9), incontinence (P = 0.3), erectile dysfunction (P = 0.9) or recto-urethral fistula between the patients treated using the standard and compression methods. CONCLUSIONS: Intraoperative transrectal compression suppresses intraoperative increase in the prostate volume and intraprostatic point shift during high-intensity focused ultrasound, having the potential to achieve precise whole-gland and lesion-targeted focal therapy.

Three-dimensional navigation system integrating position-tracking technology with a movable tablet display for percutaneous targeting.

OBJECTIVES: To assess the feasibility of a novel percutaneous navigation system (Translucent Medical, Inc., Santa Cruz, CA, USA) that integrates position-tracking technology with a movable tablet display. MATERIALS AND METHODS: A total of 18 fiducial markers, which served as the target centres for the virtual tumours (target fiducials), were implanted in the prostate and kidney of a fresh cadaver, and preoperative computed tomography (CT) was performed to allow three-dimensional model reconstruction of the surgical regions, which were registered on the body intra-operatively. The position of the movable tablet's display could be selected to obtain the best recognition of the interior anatomy. The system was used to navigate the puncture needle (with position-tracking sensor attached) using a colour-coded, predictive puncture-line. When the operator punctured the target fiducial, another fiducial, serving as the centre of the ablative treatment (treatment fiducial), was placed. Postoperative CT was performed to assess the digitized distance (representing the real distance) between the target and treatment fiducials to evaluate the accuracy of the procedure. RESULTS: The movable tablet display, with position-tracking sensor attached, enabled the surgeon to visualize the three-dimensional anatomy of the internal organs with the help of an overlaid puncture line for the puncture needle, which also had a position-tracking sensor attached. The mean (virtual) distance from the needle tip to the target (calculated using the computer workstation), was 2.5 mm. In an analysis of each digitalized axial component, the errors were significantly greater along the z-axis (P < 0.01), suggesting that the errors were caused by organ shift or deformation. CONCLUSION: This virtual navigation system, integrating a position-tracking sensor with a movable tablet display, is a promising advancement for facilitating percutaneous interventions. The movable display over the patient shows a preoperative three-dimensional image that is aligned to the patient. Moving the display moves the image, creating the feeling of looking through a window into the patient, resulting in instant perception and a direct, intuitive connection between the physician and the anatomy.

Time-dependent change of blood flow in the prostate treated with high-intensity focused ultrasound.

Avascular areas on contrast-enhanced magnetic resonance imaging have been considered to be areas of localized prostate cancer successfully treated by high-intensity focused ultrasound. However, the optimal timing of magnetic resonance imaging has not been discussed. The thermal effect of high-intensity focused ultrasound is degraded by regional prostatic blood flow. Conversely, the mechanical effect of high-intensity focused ultrasound (cavitation) is not affected by blood flow, and can induce vessel damage. In this series, the longitudinal change of blood flow on contrast-enhanced magnetic resonance imaging was observed from postoperative day 1 to postoperative day 14 in 10 patients treated with high-intensity focused ultrasound. The median rates of increase in the non-enhanced volume of the whole gland, transition zone and peripheral zone from postoperative day 1 to postoperative day 14 were 36%, 39%, and 34%, respectively. In another pathological analysis of the prostate tissue of 17 patients immediately after high-intensity focused ultrasound without neoadjuvant hormonal therapy, we observed diffuse coagulative degeneration and partial non-coagulative prostate tissue around arteries with vascular endothelial cell detachment. These observations on contrast-enhanced magnetic resonance imaging support a time-dependent change of the blood flow in the prostate treated with high-intensity focused ultrasound. Additionally, our pathological findings support the longitudinal changes of these magnetic resonance imaging findings. Further large-scale studies will investigate the most appropriate timing of contrast-enhanced magnetic resonance imaging for evaluation of the effectiveness of high-intensity focused ultrasound for localized prostate cancer.