Engineering Telodendrimer Nanocarriers for Monomeric Amphotericin B Delivery.

Systemic fungal infections are an increasingly prevalent health problem. Amphotericin B (AmB), a hydrophobic polyene antibiotic, remains the drug of choice for life-threatening invasive fungal infections. However, it has dose-limiting side effects, including nephrotoxicity. The efficacy and toxicity of AmB are directly related to its aggregation state. Here, we report the preparation of a series of telodendrimer (TD) nanocarriers with the freely engineered core structures for AmB encapsulation to fine-tune AmB aggregation status. The reduced aggregation status correlates well with the optimized antifungal activity, attenuated hemolytic properties, and reduced cytotoxicity to mammalian cells. The optimized TD nanocarrier for monomeric AmB encapsulation significantly increases the therapeutic index, reduces the in vivo toxicity, and enhances antifungal effects in mouse models with Candida albicans infection in comparison to two common clinical formulations, i.e., Fungizone and AmBisome.

Engineering Nanotrap Hydrogel for Immune Modulation in Wound Healing.

Imbalanced immune regulation leads to the abnormal wound healing process, e.g., chronic unhealing wound or hypertrophic scar formation. Thus, the attenuation of the overflowing inflammatory factors is a viable approach to maintain the homeostatic immune regulation to facilitate normal wound healing. A versatile telodendrimer (TD) nanotrap (NT) platform is developed for efficient biomolecular protein binding. The conjugation of TD NT in size-exclusive biocompatible hydrogel resin allows for topical application for cytokine scavenging. Fine-tuning the TD NT density/valency in hydrogel resin controls resin swelling, optimizes molecular diffusion, and improves cytokine capture for effective immune modulation. The hydrogel with reduced TD NT density allows for higher protein/cytokine adsorption capacity with faster kinetics, due to the reduced barrier of TD NT nano-assembly. The positively charged TD NT hydrogel exhibits superior removal of negatively charged proinflammatory cytokines from the lipopolysaccharide (LPS, a potent endotoxin) primed immune cell culture medium. The negatively charged TD NT hydrogel removes positively charged anti-inflammatory cytokines efficiently from cell culture medium. TD NT hydrogel effectively constrains the local inflammation induced by subcutaneous LPS injection in mice. These results indicate the great potential applications of the engineered TD NT hydrogel as topical immune modulatory treatments to attenuate local inflammation.

Telodendrimer-Based Macromolecular Drug Design using 1,3-Dipolar Cycloaddition for Applications in Biology.

An architectural polymer containing hydrophobic isoxazole-based dendron and hydrophilic polyethylene glycol linear tail is prepared by a combination of the robust ZnCl2 catalyzed alkyne-nitrile oxide 1,3-dipolar cycloaddition and esterification chemistry. This water soluble amphiphilic telodendrimer acts as a macromolecular biologically active agent and shows concentration dependent reduction of glioblastoma (U251) cell survival.

Telodendrimers: Promising Architectural Polymers for Drug Delivery.

Architectural complexity has played a key role in enhancing the efficacy of nanocarriers for a variety of applications, including those in the biomedical field. With the continued evolution in designing macromolecules-based nanoparticles for drug delivery, the combination approach of using important features of linear polymers with dendrimers has offered an advantageous and viable platform. Such nanostructures, which are commonly referred to as telodendrimers, are hybrids of linear polymers covalently linked with different dendrimer generations and backbones. There is considerable variety in selection from widely studied linear polymers and dendrimers, which can help tune the overall composition of the resulting hybrid structures. This review highlights the advances in articulating syntheses of these macromolecules, and the contributions these are making in facilitating therapeutic administration. Limited progress has been made in the design and synthesis of these hybrid macromolecules, and it is through an understanding of their physicochemical properties and aqueous self-assembly that one can expect to fully exploit their potential in drug delivery.

Unique Photochemo-Immuno-Nanoplatform against Orthotopic Xenograft Oral Cancer and Metastatic Syngeneic Breast Cancer.

Sophisticated self-assembly may endow materials with a variety of unique functions that are highly desirable for therapeutic nanoplatform. Herein, we report the coassembly of two structurally defined telodendrimers, each comprised of hydrophilic linear PEG and hydrophobic cholic acid cluster as a basic amphiphilic molecular subunit. One telodendrimer has four added indocyanine green derivatives, leading to excellent photothermal properties; the other telodendrimer has four sulfhydryl groups designed for efficient intersubunit cross-linking, contributing to superior stability during circulation. The coassembled nanoparticle (CPCI-NP) possesses superior photothermal conversion efficiency as well as efficient encapsulation and controlled release of cytotoxic molecules and immunomodulatory agents. CPCI-NP loaded with doxorubicin has proven to be a highly efficacious combination photothermal/chemotherapeutic nanoplatform against orthotopic OSC-3 oral cancer xenograft model. When loaded with imiquimod, a potent small molecule immunostimulant, CPCI-NP is found to be highly effective against 4T1 syngeneic murine breast cancer model, particularly when photothermal/immuno-therapy is given in combination with PD-1 checkpoint blockade antibody. Such triple therapy not only eradicates the light-irradiated primary tumors, but also activates systemic antitumor immunoactivity, causing tumor death at light-unexposed distant tumor sites. This coassembled multifunctional, versatile, and easily scalable photothermal immuno-nanoplatform shows great promise for clinical translation.

Cell-free production of a functional oligomeric form of a Chlamydia major outer-membrane protein (MOMP) for vaccine development.

Chlamydia is a prevalent sexually transmitted disease that infects more than 100 million people worldwide. Although most individuals infected with Chlamydia trachomatis are initially asymptomatic, symptoms can arise if left undiagnosed. Long-term infection can result in debilitating conditions such as pelvic inflammatory disease, infertility, and blindness. Chlamydia infection, therefore, constitutes a significant public health threat, underscoring the need for a Chlamydia-specific vaccine. Chlamydia strains express a major outer-membrane protein (MOMP) that has been shown to be an effective vaccine antigen. However, approaches to produce a functional recombinant MOMP protein for vaccine development are limited by poor solubility, low yield, and protein misfolding. Here, we used an Escherichia coli-based cell-free system to express a MOMP protein from the mouse-specific species Chlamydia muridarum (MoPn-MOMP or mMOMP). The codon-optimized mMOMP gene was co-translated with Δ49apolipoprotein A1 (Δ49ApoA1), a truncated version of mouse ApoA1 in which the N-terminal 49 amino acids were removed. This co-translation process produced mMOMP supported within a telodendrimer nanolipoprotein particle (mMOMP-tNLP). The cell-free expressed mMOMP-tNLPs contain mMOMP multimers similar to the native MOMP protein. This cell-free process produced on average 1.5 mg of purified, water-soluble mMOMP-tNLP complex in a 1-ml cell-free reaction. The mMOMP-tNLP particle also accommodated the co-localization of CpG oligodeoxynucleotide 1826, a single-stranded synthetic DNA adjuvant, eliciting an enhanced humoral immune response in vaccinated mice. Using our mMOMP-tNLP formulation, we demonstrate a unique approach to solubilizing and administering membrane-bound proteins for future vaccine development. This method can be applied to other previously difficult-to-obtain antigens while maintaining full functionality and immunogenicity.

Telodendrimer functionalized hydrogel platform for sustained antibiotics release in infection control.

Wound infection commonly causes delayed healing, especially in the setting of chronic wounds. Local release of antibiotics is considered a viable approach to treat chronic wounds. We have developed a versatile telodendrimer (TD) platform for efficient loading of charged antibiotic molecules via a combination of multivalent and synergistic charge and hydrophobic interactions. The conjugation of TD in biocompatible hydrogel allows for topical application to provide sustained antibiotic release. Notably, a drug loading capacity as high as 20 % of the drug-to-resin dry weight ratio can be achieved. The payload content (PC) and release profile of the various antibiotics can be optimized by fine-tuning TD density and valency in hydrogel based on the charge and hydrophobic features of the drug, e.g., polymyxin B (PMB), gentamycin (GM), and daptomycin (Dap), for effective infection control. We have shown that hydrogel with moderately reduced TD density demonstrates a more favorable release profile than hydrogel with higher TD density. Antibiotics loaded in TD hydrogel have comparable antimicrobial potency and reduced cytotoxicity compared to the free antibiotics due to a prolonged, controlled drug release profile. In a mouse model of skin and soft tissue infection, the subcutaneous administration of PMB-loaded TD hydrogel effectively eliminated the bacterial burden. Overall, these results suggest that engineerable TD hydrogels have great potential as a topical treatment to control infection for wound healing. STATEMENT OF SIGNIFICANCE: Wound infection causes a significant delay in the wound healing process, which results in a significant financial and resource burden to the healthcare system. PEGA-telodendrimer (TD) resin hydrogel is an innovative and versatile platform that can be fine-tuned to efficiently encapsulate different antibiotics by altering charged and hydrophobic structural moieties. Additionally, this platform is advantageous as the TD density in the resin can also be fine-tuned to provide the desired antibiotic payload release profile. Sustained antibiotics release through optimization of TD density provides a prolonged therapeutic window and reduces burst release-induced cytotoxicity compared to conventional antibiotics application. Studies in a preclinical mouse model of bacteria-induced skin and soft tissue infection demonstrated promising therapeutic efficacy as evidenced by effective infection control and prolonged antibacterial efficacy of antibiotics-loaded PEGA-TD resin. In conclusion, the PEGA-TD resin platform provides a highly customizable approach for effective antibiotics release with significant potential for topical application to treat various bacterial wound infections to promote wound healing.

"Click" amphotericin B in prodrug nanoformulations for enhanced systemic fungemia treatment.

Severe nephrotoxicity and infusion-related side effects pose significant obstacles to the clinical application of Amphotericin B (AmB) in life-threatening systemic fungal infections. In pursuit of a cost-effective and safe formulation, we have introduced multiple phenylboronic acid (PBA) moieties onto a linear dendritic telodendrimer (TD) scaffold, enabling effective AmB conjugation via boronate chemistry through a rapid, high yield, catalysis-free and dialysis-free "Click" drug loading process. Optimized AmB-TD prodrugs self-assemble into monodispersed micelles characterized by small particle sizes and neutral surface charges. AmB prodrugs sustain drug release in circulation, which is accelerated in response to the acidic pH and Reactive Oxygen Species (ROS) in the infection and inflammation. Prodrugs mitigate the AmB aggregation status, reduce cytotoxicity and hemolytic activity compared to Fungizone®, and demonstrate superior antifungal activity to AmBisome®. AmB-PEG5kBA4 has a comparable maximum tolerated dose (MTD) to AmBisome®, while over 20-fold increase than Fungizone®. A single dose of AmB-PEG5kBA4 demonstrates superior efficacy to Fungizone® and AmBisome® in treating systemic fungal infections in both immunocompetent and immunocompromised mice.

Functionalized core-shell nanogel scavenger for immune modulation therapy in sepsis.

Sepsis is a complex, life-threatening hyperinflammatory syndrome associated with organ failure and high mortality due to lack of effective treatment options. Here we report a core-shell hydrogel nanoparticle with the core functionalized with telodendrimer (TD) nanotrap (NT) to control hyperinflammation in sepsis. The combination of multi-valent charged and hydrophobic moieties in TD enables effective binding with biomolecules in NT. The higher crosslinking in the shell structure of nanogel excludes the abundant large serum proteins and allows for size-selectivity in scavenging the medium-sized septic molecules (10-30 kDa), e.g., lipopolysaccharides (LPS, a potent endotoxin in sepsis), thus reducing cytokine production. At the same time, the core-shell TD NT nanogel captures the over-flowing proinflammatory cytokines effectively both in vitro and in vivo from biological fluids to further control hyperinflammation. Intraperitoneal injection of core-shell TD NT nanogel effectively attenuates NF-κB activation and cytokine production in LPS-induced septic mouse models. These results indicate the potential applications of the injectable TD NT core-shell nanogel to attenuate local or systemic inflammation.

Synergistic effect of the anti-PD-1 antibody with blood stable and reduction sensitive curcumin micelles on colon cancer.

Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) is a potent anticancer drug with versatile biological activities, while the clinical translation of curcumin is severely limited due to its hydrophobicity, rapid elimination, and metabolism in the blood circulation. Herein, we aim to unravel the potential of curcumin as a synergistic agent with immunotherapy in the treatment of cancers. In an effort to minimize premature release and improve the systemic bioavailability, a superior blood stable and reduction sensitive curcumin micellar formulation, of which the release can be triggered by cancer cells, is rationally designed. We have synthesized a telodendrimer (mPEG-PLA-(LA)4) capable of forming reversible disulfide crosslinked micelles (DCMs). The curcumin loaded DCMs (Cur/DCMs) are spherical with a uniform size of 24.6 nm. The in vitro release profile demonstrates that curcumin releases significantly slower from DCMs than that from non-crosslinked micelles (NCMs), while the release can be accelerated with the increasing concentration of reducing agent glutathione (GSH). Intravenous administration of Cur/DCMs stably retains curcumin in the bloodstream and efficiently improves the systemic bioavailability. Furthermore, Cur/DCMs exhibit synergistic anticancer efficacy when combined with the anti-PD-1 antibody in an MC-38 colon cancer xenograft model. Our results potentiate the integration of blood stable curcumin nanoformulation and immunotherapy for cancer treatment.

A Rationally Designed Micellar Nanocarrier for the Delivery of Hydrophilic Methotrexate in Psoriasis Treatment.

Methotrexate (MTX) is broadly applied in the clinic for the treatments of cancers and autoimmune diseases. Targeted delivery of MTX is attractive to improve its efficacy and reduce off-target toxicity. However, MTX encapsulation in nanoparticle is challenging due to its high water solubility. We rationally designed a well-defined telodendrimer (TD) nanocarrier based on MTX structure to sequester it in nanoparticles. Riboflavin (Rf) and positive charges groups were precisely conjugated on TD to form multivalent hydrogen bonds, π-π stacking and electrostatic interactions with MTX. A reverse micelle approach was developed to preset MTX and TD interactions in the core of micelles, which ensures the effective MTX loading upon dispersion into aqueous solution. As results, MTX loading capacity reaches over 20% (w/w) in the optimized nanocarrier with the particle size of 20-30 nm. The nanoformulations sustain the release of MTX in a controlled manner and exhibit excellent hemocompatibility. The in vitro cellular uptake of MTX was significantly improved by the nanoformulations. The potency of MTX nanoformulations is comparable to the free MTX in cytotoxicity. A psoriasis-like skin inflammation model was induced in mouse by imiquimod (IMQ) stimulation. MTX nanoformulations improved the psoriasis targeting and exhibited a superior long-lasting efficacy in reducing skin inflammation compared with the free MTX in psoriasis treatment.

Polycation-telodendrimer nanocomplexes for intracellular protein delivery.

Intracellular delivery of protein therapeutics by cationic polymer vehicles is an emerging technique that is, however, encountering poor stability, high cytotoxicity and non-specific cell uptake. Herein, we present a facile strategy to optimize the protein-polycation complexes by encapsulating with linear-dendritic telodendrimers. The telodendrimers with well-defined structures enable the rational design and integration of multiple functionalities for efficient encapsulation of the protein-polycation complexes by multivalent and hybrid supramolecular interactions to produce sub-20 nm nanoparticles. This strategy not only reduces the polycation-associated cytotoxicity and hemolytic activity, but also eliminates the aggregation and non-specific binding of polycations to other biomacromolecules. Moreover, the telodendrimers dissociate readily from the complexes during the cellular uptake process, which restores the capability of polycations for intracellular protein delivery. This strategy overcomes the limitations of polycationic vectors for intracellular delivery of protein therapeutics.

Cholic acid-based novel micellar nanoplatform for delivering FDA-approved taxanes.

AIM: To structurally modify our existing cholic acid (CA)-based telodendrimer (TD; PEG5K-CA8) for effective micellar nanoencapsulation and delivery of the US FDA-approved members of taxane family. MATERIALS & METHODS: Generation of hybrid TDs was achieved by replacing four of the eight CAs with biocompatible organic moieties using solution-phase peptide synthesis. Drug loading was done using the standard evaporation method. RESULTS: Hybrid TDs can generate micelles with narrow size distributions, low critical micelle concentration values (1-6 µM), better hematocompatibility and lack of in vitro cytotoxicity. CONCLUSION: Along with PEG5K-CA8, CA-based hybrid nanoplatform is the first of its kind that can stably encapsulate all three FDA-approved taxanes with nearly 100% efficiency up to 20% (w/w) loading.

Riboflavin-containing telodendrimer nanocarriers for efficient doxorubicin delivery: High loading capacity, increased stability, and improved anticancer efficacy.

We have developed two linear-dendritic telodendrimers (TDs) with rational design using amphiphilic riboflavin (Rf) as building blocks for efficient doxorubicin (DOX) delivery. Micellar TD nanoparticles (NPs) are composed of a hydrophilic polyethylene glycol (PEG) shell and a Rf-containing affinitive core for DOX encapsulation. Strong DOX-Rf interactions and amphiphilic Rf structure render these nanocarriers with an ultra-high DOX loading capacity (>1/1, DOX/TD, w/w), ∼100% loading efficiency, the sustained drug release and the optimal particle sizes (20-40 nm) for efficient tumor-targeted drug delivery. These nanoformulations significantly prolonged DOX circulation time in the blood without the accelerated clearance observed after multiple injections. DOX-TDs target several types of tumors efficiently in vivo, e.g. Raji lymphoma, MDA-MB-231 breast cancer, and SKOV-3 ovarian cancer. In vivo maximum tolerated dose (MTD) of DOX was increased by 2-2.5 folds for the nanoformulations in mice relative to those of free DOX and Doxil®. These nanoformulations significantly inhibited tumor growth and prolonged survival of mice bearing SKOV-3 ovarian cancer xenografts. In summary, Rf-containing nanoformulations with high DOX loading capacity, improved stability and efficient tumor targeting lead to superior antitumor efficacy, which merit the further development for clinical application.

Ratiometric drug delivery using non-liposomal nanocarriers as an approach to increase efficacy and safety of combination chemotherapy.

The observation that different drug ratios of the same drug combination can lead to synergistic or antagonistic effects when tested against the same cancer cell line in vitro gave rise to a new trend, the ratiometric delivery. This strategy consists of co-encapsulating a specific synergistic ratio of a drug combination into a nanocarrier so that synergism observed in vitro will be faithfully translated to in vivo, optimizing combination therapy. In this review we focus on how to quantify synergism in vitro, followed by how this affected the evolution of nanocarriers culminating in the ratiometric delivery, and finally we summarize the results of the non-liposomal formulations that were built upon this concept.

Controlling the diameter, monodispersity, and solubility of ApoA1 nanolipoprotein particles using telodendrimer chemistry.

Nanolipoprotein particles (NLPs) are nanometer-scale discoidal particles that feature a phospholipid bilayer confined within an apolipoprotein "scaffold," which are useful for solubilizing hydrophobic molecules such as drugs and membrane proteins. NLPs are synthesized either by mixing the purified apolipoprotein with phospholipids and other cofactors or by cell-free protein synthesis followed by self-assembly of the nanoparticles in the reaction mixture. Either method can be problematic regarding the production of homogeneous and monodispersed populations of NLPs, which also currently requires multiple synthesis and purification steps. Telodendrimers (TD) are branched polymers made up of a dendritic oligo-lysine core that is conjugated to linear polyethylene glycol (PEG) on one end, and the lysine "branches" are terminated with cholic acid moieties that enable the formation of nanomicelles in aqueous solution. We report herein that the addition of TD during cell-free synthesis of NLPs produces unique hybrid nanoparticles that have drastically reduced polydispersity as compared to NLPs made in the absence of TD. This finding was supported by dynamic light scattering, fluorescence correlation spectroscopy, and cryo transmission electron microscopy (Cryo-EM). These techniques demonstrate the ability of TDs to modulate both the NLP size (6-30 nm) and polydispersity. The telodendrimer NLPs (TD-NLPs) also showed 80% less aggregation as compared to NLPs alone. Furthermore, the versatility of these novel nanoparticles was shown through direct conjugation of small molecules such as fluorescent dyes directly to the TD as well as the insertion of a functional membrane protein.

Biodistribution and pharmacokinetics of a telodendrimer micellar paclitaxel nanoformulation in a mouse xenograft model of ovarian cancer.

BACKGROUND: A multifunctional telodendrimer-based micelle system was characterized for delivery of imaging and chemotherapy agents to mouse tumor xenografts. Previous optical imaging studies demonstrated qualitatively that these classes of nanoparticles, called nanomicelles, preferentially accumulate at tumor sites in mice. The research reported herein describes the detailed quantitative imaging and biodistribution profiling of nanomicelles loaded with a cargo of paclitaxel. METHODS: The telodendrimer was covalently labeled with ¹²5I and the nanomicelles were loaded with ¹4C-paclitaxel, which allowed measurement of pharmacokinetics and biodistribution in the mice using microSPECT/CT imaging and liquid scintillation counting, respectively. RESULTS: The radio imaging data showed preferential accumulation of nanomicelles at the tumor site along with a slower clearance rate than paclitaxel formulated in Cremophor EL (Taxol®). Liquid scintillation counting confirmed that ¹4C-labeled paclitaxel sequestered in nanomicelles had increased uptake by tumor tissue and slower pharmacokinetics than Taxol. CONCLUSION: Overall, the results indicate that nanomicelle-formulated paclitaxel is a potentially superior formulation compared with Taxol in terms of water solubility, pharmacokinetics, and tumor accumulation, and may be clinically useful for both tumor imaging and improved chemotherapy applications.