Injectable hydrogel based on liposome self-assembly for controlled release of small hydrophilic molecules.

Controlled release of low molecular weight hydrophilic drugs, administered locally, allows maintenance of high concentrations at the target site, reduces systemic side effects, and improves patient compliance. Injectable hydrogels are commonly used as a vehicle. However, slow release of low molecular weight hydrophilic drugs is very difficult to achieve, mainly due to a rapid diffusion of the drug out of the drug delivery system. Here we present an injectable and self-healing hydrogel based entirely on the self-assembly of liposomes. Gelation of liposomes, without damaging their structural integrity, was induced by modifying the cholesterol content and surface charge. The small hydrophilic molecule, sodium fluorescein, was loaded either within the extra-liposomal space or encapsulated into the aqueous cores of the liposomes. This encapsulation strategy enabled the achievement of controlled and adjustable release profiles, dependent on the mechanical strength of the gel. The hydrogel had a high mechanical strength, minimal swelling, and slow degradation. The liposome-based hydrogel had prolonged mechanical stability in vivo with benign tissue reaction. This work presents a new class of injectable hydrogel that holds promise as a versatile drug delivery system. STATEMENT OF SIGNIFICANCE: The porous nature of hydrogels poses a challenge for delivering small hydrophilic drug, often resulting in initial burst release and shorten duration of release. This issue is particularly pronounced with physically crosslinked hydrogels, since their matrix can swell and dissipate rapidly, but even in cases where the polymers in the hydrogel are covalently cross-linked, small molecules can be rapidly released through its porous mesh. Here we present an injectable self-healing hydrogel based entirely on the self-assembly of liposomes. Small hydrophilic molecules were entrapped inside the extra-liposomal space or loaded into the aqueous cores of the liposomes, allowing controlled and tunable release profiles.

Long-Circulating Vasoactive 1,18-Octadecanedioic Acid-Terlipressin Conjugate.

Hepatorenal syndrome (HRS) is a life-threatening complication of end-stage liver disease first reported over a century ago, but its management still poses an unmet challenge. A therapeutic agent found to stabilize the condition is a short cyclic peptide, vasopressin analogue, terlipressin (TP). While TP is commonly prescribed for HRS patients in most parts of the world, it was only recently approved for use in the United States. TP exhibits short circulation half-lives and adverse side effects associated with the dose required. Herein, we present a 1,18-octadecanedioic acid (ODDA) conjugate of the cyclic peptide (ODDA-TP), which enables noncovalent binding to serum albumin via native fatty acid binding modes. ODDA-TP is demonstrated to outperform TP alone in studies including in vitro cellular receptor activation, stability in plasma, pharmacokinetics, and performance in vivo in rats. Specifically, ODDA-TP had an elimination half-life 20 times that of TP alone while exhibiting a superior safety profile.

A hybrid nanoparticle-protein hydrogel system for prolonged local anesthesia.

Local anesthetics are effective in relieving pain, but their duration of action is short. Therefore, the development of injectable sustained release systems to prolong the effect of local anesthetics has been of interest. In such systems delivering conventional local anesthetics, it has been challenging to achieve long durations of effect, particularly without incurring tissue toxicity. To overcome these challenges, we created a platform comprising a protein hydrogel incorporating hydrophobic local anesthetic (bupivacaine) nanoparticles. The nanoparticles were prepared by anti-solvent precipitation stabilized with bovine serum albumin (BSA), followed by crosslinking with glutaraldehyde (GA). The resulting BSA hydrogels prolonged release of bupivacaine in vitro. When bupivacaine nanoparticles within crosslinked BSA were injected at the sciatic nerve in rats, a duration of nerve block of 39.9 h was obtained, compared to 5.5 h for the commercial bupivacaine liposome suspension EXPAREL®. Tissue reaction was benign. We further demonstrated that this system could control the release of the amphiphilic drug diphenhydramine and the hydrophobic paclitaxel.

Thrombospondin-1 proteomimetic polymers exhibit anti-angiogenic activity in a neovascular age-related macular degeneration mouse model.

Neovascular age-related macular degeneration (nAMD) is the leading cause of blindness in the developed world. Current therapy includes monthly intraocular injections of anti-VEGF antibodies, which are ineffective in up to one third of patients. Thrombospondin-1 (TSP1) inhibits angiogenesis via CD36 binding, and its down-regulated expression is negatively associated with the onset of nAMD. Here, we describe TSP1 mimetic protein-like polymers (TSP1 PLPs). TSP1 PLPs bind CD36 with high affinity, resist degradation, show prolonged intraocular half-lives (13.1 hours), have no toxicity at relevant concentrations in vivo (40 µM), and are more efficacious in ex vivo choroidal sprouting assays compared to the peptide sequence and Eylea (aflibercept), the current standard of care anti-VEGF treatment. Furthermore, PLPs exhibit superior in vivo efficacy in a mouse model for nAMD compared to control PLPs consisting of scrambled peptide sequences, using fluorescein angiography and immunofluorescence. Since TSP-1 inhibits angiogenesis by VEGF-dependent and independent mechanisms, TSP1 PLPs are a potential therapeutic for patients with anti-VEGF treatment-resistant nAMD.

Origin of Proteolytic Stability of Peptide-Brush Polymers as Globular Proteomimetics.

Peptide-brush polymers (PBPs), wherein every side-chain of the polymers is peptidic, represent a new class of proteomimetic with unusually high proteolytic resistance while maintaining bioactivity. Here, we sought to determine the origin of this behavior and to assess its generality via a combined theory and experimental approach. A series of PBPs with various polymer backbone structures were prepared and examined for their proteolytic stability and bioactivity. We discovered that an increase in the hydrophobicity of the polymer backbones is predictive of an elevation in proteolytic stability of the side-chain peptides. Computer simulations, together with small-angle X-ray scattering (SAXS) analysis, revealed globular morphologies for these polymers, in which pendant peptides condense around hydrophobic synthetic polymer backbones driven by the hydrophobic effect. As the hydrophobicity of the polymer backbones increases, the extent of solvent exposure of peptide cleavage sites decreases, reducing their accessibility to proteolytic enzymes. This study provides insight into the important factors driving PBP aqueous-phase structures to behave as globular, synthetic polymer-based proteomimetics.

pH-Responsive Charge-Conversion Progelator Peptides.

A simple strategy for generating stimuli-responsive peptide-based hydrogels via charge-conversion of a self-assembling peptide (SAP) is described. These materials are formulated as soluble, polyanionic peptides, containing maleic acid, citraconic acid, or dimethylmaleic acid masking groups on each lysine residue, which do not form assemblies, but instead flow easily through high gauge needles and catheters. Acid-induced mask hydrolysis renews the zwitterionic nature of the peptides with concomitant and rapid self-assembly via ß-sheet formation into rehealable hydrogels. The use of different masks enables one to tune pH responsiveness and assembly kinetics. In anticipation of their potential for in vivo hydrogel delivery and use, progelators exhibit hemocompatibility in whole human blood, and their peptide components are shown to be noncytotoxic. Finally, demonstration of stimuli-induced self-assembly for dye sequestration suggests a simple, non-covalent strategy for small molecule encapsulation in a degradable scaffold. In summary, this simple, scalable masking strategy allows for preparation of responsive, dynamic self-assembling biomaterials. This work sets the stage for implementing biodegradable therapeutic hydrogels that assemble in response to physiological, disease-relevant states of acidosis.

High efficiency loading of micellar nanoparticles with a light switch for enzyme-induced rapid release of cargo.

Polymeric nanoscale materials able to target and accumulate in the tumor microenvironment (TME) offer promising routes for a safer delivery of anticancer drugs. By reaching their targets before significant amounts of drug are released, such materials can reduce off-target side effects and maximize drug concentration in the TME. However, poor drug loading capacity and inefficient nanomaterial penetration into the tumor can limit their therapeutic efficacy. Herein, we provide a novel approach to achieve high loading profiles while ensuring fast and efficient drug penetration in the tumor. This is achieved by co-polymerizing light-sensitive paclitaxel with monomers responsive to tumor-associated enzymes, and assembling the resulting di-block copolymers into spherical micelles. While light exposure enables paclitaxel to decouple from the polymeric backbone into light-activated micelles, enzymatic digestion in the TME initiates its burst release. Through a series of in vitro cytotoxicity assays, we demonstrate that these light-switch micelles hold greater potency than covalently linked, non-triggered micelles, and enable therapeutic profiles comparable to that of the free drug.

Proapoptotic Peptide Brush Polymer Nanoparticles via Photoinitiated Polymerization-Induced Self-Assembly.

Herein, we report the photoinitiated polymerization-induced self-assembly (photo-PISA) of spherical micelles consisting of proapoptotic peptide-polymer amphiphiles. The one-pot synthetic approach yielded micellar nanoparticles at high concentrations and at scale (150 mg mL-1 ) with tunable peptide loadings up to 48 wt. %. The size of the micellar nanoparticles was tuned by varying the lengths of hydrophobic and hydrophilic building blocks. Critically, the peptide-functionalized nanoparticles imbued the proapoptotic "KLA" peptides (amino acid sequence: KLAKLAKKLAKLAK) with two key properties otherwise not inherent to the sequence: 1) proteolytic resistance compared to the oligopeptide alone; 2) significantly enhanced cell uptake by multivalent display of KLA peptide brushes. The result was demonstrated improved apoptosis efficiency in HeLa cells. These results highlight the potential of photo-PISA in the large-scale synthesis of functional, proteolytically resistant peptide-polymer conjugates for intracellular delivery.

Biomolecular Densely Grafted Brush Polymers: Oligonucleotides, Oligosaccharides and Oligopeptides.

In this Minireview, we describe synthetic polymers densely functionalized with sequence-defined biomolecular sidechains. We focus on synthetic brush polymers of oligonucleotides, oligosaccharides, and oligopeptides, prepared via graft-through polymerization from biomolecule functionalized monomers. The resulting structures are brush polymers wherein a biomolecular graft is positioned at each monomer backbone unit. We describe key synthetic milestones, identify synthetic opportunities, and highlight recent advances in the field, including biological applications.

Bioactive Peptide Brush Polymers via Photoinduced Reversible-Deactivation Radical Polymerization.

Harnessing metal-free photoinduced reversible-deactivation radical polymerization (photo-RDRP) in organic and aqueous phases, we report a synthetic approach to enzyme-responsive and pro-apoptotic peptide brush polymers. Thermolysin-responsive peptide-based polymeric amphiphiles assembled into spherical micellar nanoparticles that undergo a morphology transition to worm-like micelles upon enzyme-triggered cleavage of coronal peptide sidechains. Moreover, pro-apoptotic polypeptide brushes show enhanced cell uptake over individual peptide chains of the same sequence, resulting in a significant increase in cytotoxicity to cancer cells. Critically, increased grafting density of pro-apoptotic peptides on brush polymers correlates with increased uptake efficiency and concurrently, cytotoxicity. The mild synthetic conditions afforded by photo-RDRP, make it possible to access well-defined peptide-based polymer bioconjugate structures with tunable bioactivity.

Recent Advances in Amphiphilic Polymer-Oligonucleotide Nanomaterials via Living/Controlled Polymerization Technologies.

Over the past decade, the field of polymer-oligonucleotide nanomaterials has flourished because of the development of synthetic techniques, particularly living polymerization technologies, which provide access to polymers with well-defined architectures, precise molecular weights, and terminal or side-chain functionalities. Various "living" polymerization methods have empowered chemists with the ability to prepare functional polymer-oligonucleotide conjugates yielding a library of architectures, including linear diblock, comb, star, hyperbranched star, and gel morphologies. Since oligonucleotides are hydrophilic and synthetic polymers can be tailored with hydrophobicity, these amphiphilic polymer-oligonucleotide conjugates are capable of self-assembling into nanostructures with different shapes, leading to many high-value-added biomedical applications, such as drug delivery systems, gene regulation, and 3D-bioprinting. This review aims to highlight the main living polymerization approaches to polymer-oligonucleotide conjugates, including ring-opening metathesis polymerization, atom transfer radical polymerization (ATRP), reversible addition-fragmentation transfer polymerization (RAFT), and ring-opening polymerization of cyclic esters and N-carboxyanhydride. The self-assembly properties and resulting applications of polymer-DNA hybrid materials are highlighted as well.

Mapping Exchangeable Protons to Monitor Protein Alterations in the Brain of an Alzheimer's Disease Mouse Model by Using MRI.

OBJECTIVE: The study aimed to investigate exchangeable proton signals of Aß proteins of the brains of Alzheimer's disease (AD) model mice by using a chemical exchange-sensitive spin-lock (CESL) MR imaging technique. METHOD: Eight non-transgenic (Tg) mice (5 young and 3 old) and twelve Tg-APPswe/PSdE9 mice (5 young and 7 old) were used in this study. CESL Z-spectra were obtained by using two saturation powers, which were ω1 = 25 Hz with TSL = 3.0 s and ω1 = 500 Hz with TSL = 150 ms, at 71 offsets with uneven intervals between the offset frequencies at Ω = ±7.0 ppm at a 9.4-T animal MRI system. For Zspectrum analyses, regions of interest (ROIs) were drawn in the cortex, hippocampus, and thalamus of both hemispheres. Magnetization transfer ratio asymmetry (MTRasym) curves were obtained from the Zspectra. The Mann-Whitney test was used to compare the MTRasym values between the Tg and non-Tg mice for each offset frequency and for each ROI. RESULTS: The water saturation width of the full Z-spectrum was narrow with the 25-Hz saturation power, but relatively broad with the 500-Hz saturation power. With the 25-Hz CESL saturation power, most of the MTRasym values were negative for 3.5-, 3.0-, 2.0-, and 0.9-ppm offset frequencies and the MTRasym values were significantly different between the control and Tg groups only in the left thalamus region at 3.5 ppm offset (p=0.0487). The MTRasym values were -6% to -7% for both 3.5 and 3.0 ppm, but less than -2% for both 2.0 and 0.9 ppm. With 500-Hz CESL saturation power, all the MTRasym values were positive for the 3.5-, 3.0-, 2.0-, and 0.9-ppm offset frequencies and the MTRasym values were not significantly different between the control and Tg groups at all ROIs and at all offset frequencies. However, a trend towards a significant difference was observed between the control and Tg groups in the right cortex at 3.5 ppm (p=0.0578). The MTRasym values were 6% to 9% for 3.5, 3.0, and 2.0 ppm, but less than 2% for 0.9 ppm. CONCLUSION: In an in-vivo AD model experiment, MTRasym values increased with the high saturation power than with the low saturation power. The MTRasym values were not significantly different, except in the left thalamus region at 3.5 ppm offset. The CESL technique should be further developed to enable its application in the brain of patients with neurodegenerative diseases.

Chemical-exchange-sensitive MRI of amide, amine and NOE at 9.4 T versus 15.2 T.

Chemical exchange (CE)-sensitive MRI benefits greatly from stronger magnetic fields; however, field effects on CE-sensitive imaging have not yet been studied well in vivo. We have compared CE-sensitive Z-spectra and maps obtained at the fields of 9.4 T and 15.2 T in phantoms and rats with off-resonance chemical-exchange-sensitive spin lock (CESL), which is similar to conventional chemical exchange saturation transfer. At higher fields, the background peak at water resonance has less spread and the exchange rate relative to chemical shift decreases, thus CESL intensity is dependent on B0 . For the in vivo amide and nuclear Overhauser enhancement (NOE) composite resonances of rat brains, intensities were similar for both magnetic fields, but effective amide proton transfer and NOE values obtained with three-point quantification or a curve fitting method were larger at 15.2 T due to the reduced spread of attenuation at the direct water resonance. When using intermediate exchange-sensitive irradiation parameters, the amine proton signal was 65% higher at 15.2 T than at 9.4 T due to a reduced ratio of exchange rate to chemical shift. In summary, increasing magnetic field provides enhancements to CE-sensitive signals in the intermediate exchange regime and reduces contamination from background signals in the slow exchange regime. Consequently, ultrahigh magnetic field is advantageous for CE-sensitive MRI, especially for amine and hydroxyl protons.

A strategy for the separation of diterpenoid isomers from the root of Aralia continentalis by countercurrent chromatography: The distribution ratio as a substitute for the partition coefficient and a three-phase solvent system.

Aralia continentalis (Araliaceae) is widely used as a medicinal plant in East Asia. Previous studies have indicated that diterpenoid isomers (kaurenoic acid, continentalic acid, and ent-continentalic acid) are the major bioactive compounds of this plant. A new strategy was developed to alleviate difficulties in the separation of these isomers from this plant. A three-phase solvent system was applied to separate the isomers, and furthermore, the distribution ratio (Kc) was introduced as a substitute for the partition coefficient (KD). For compounds exhibiting a single equilibrium, their distributions in two immiscible phases were only affected by the partition coefficient of each solute. However, compounds that have a dissociating functional group (e.g., -COOH) are involved in two types of equilibrium in the two-phase system. In this case, the partitioning behaviors of the solutes are greatly affected by the pH of the solution. A mathematical prediction was applied for adjusting the solutions to the proper pH values. To prevent non-used phase (medium phase) waste, both the stationary phase (upper phase) and mobile phase (lower phase) were prepared on-demand without pre-saturation with the application of (1)H NMR. Each fraction obtained was collected and dried, yielding the following diterpenoid isomers from the 50mg injected sample: kaurenoic acid (19.7mg, yield: 39%) and ent-continentalic acid (21.3mg, yield: 42%).

Unlocking the pH-responsive degradability of fumaramic acid derivatives using photoisomerization.

Fumaramic acid derivatives can be converted into their cis isomer maleamic acid derivatives under UV illumination, and these maleamic acid derivatives show pH-responsive degradability at acidic pH only after the preceding photoisomerization. The rate of the tandem photoisomerization-degradation of fumaramic acid derivatives can be finely controlled by changing the substituents on the double bond. Photoisomerization-based unlocking of the pH-responsive degradability of fumaramic acid derivatives has strong potential for the development of multisignal-responsive smart materials in biomedical applications.

Comparison of pH-sensitive degradability of maleic acid amide derivatives.

We synthesized five maleic acid amide derivatives (maleic, citraconic, cis-aconitic, 2-(2'-carboxyethyl) maleic, 1-methyl-2-(2'-carboxyethyl) maleic acid amide), and compared their degradability for the future development of pH-sensitive biomaterials with tailored kinetics of the release of drugs, the change of charge density, and the degradation of scaffolds. The degradation kinetics was highly dependent upon the substituents on the cis-double bond. Among the maleic acid amide derivatives, 2-(2'-carboxyethyl) maleic acid amide with one carboxyethyl and one hydrogen substituent showed appropriate degradability at weakly acidic pH, and the additional carboxyl group can be used as a pH-sensitive linker.