An enzyme‐responsive, paclitaxel‐loaded nanoparticle is described and assessed in vivo in a human fibrosarcoma murine xenograft. This work represents a proof‐of‐concept study demonstrating the ...utility of enzyme‐responsive nanoscale drug carriers capable of targeted accumulation and retention in tumor tissue in response to overexpressed endogenous enzymes.
Conspectus In this Account, we describe the organization of functional peptides as densely arrayed side chains on polymer scaffolds which we introduce as a new class of material called ...poly(peptide). We describe two general classes of poly(peptide): (1) Peptide–Polymer Amphiphiles (PPAs), which consist of block copolymers with a dense grouping of peptides arrayed as the side chains of the hydrophilic block and connected to a hydrophobic block that drives micelle assembly, and (2) Protein-like Polymers (PLPs), wherein peptide-brush polymers are composed from monomers, each containing a peptide side chain. Peptides organized in this manner imbue polymers or polymeric nanoparticles with a range of functional qualities inherent to their specific sequence. Therefore, polymers or nanoparticles otherwise lacking bioactivity or responsiveness to stimuli, once linked to a peptide of choice, can now bind proteins, enter cells and tissues, have controlled and switchable biodistribution patterns, and be enzyme substrates (e.g., for kinases, phosphatases, proteases). Indeed, where peptide substrates are incorporated, kinetically or thermodynamically driven morphological transitions can be enzymatically induced in the polymeric material. Synergistically, the polymer enforces changes in peptide activity and function by virtue of packing and constraining the peptide. The scaffold can protect peptides from proteolysis, change the pharmacokinetic profile of an intravenously injected peptide, increase the cellular uptake of an otherwise cell impermeable therapeutic peptide, or change peptide substrate activity entirely. Moreover, in addition to the sequence-controlled peptides (generated by solid phase synthesis), the polymer can carry its own sequence-dependent information, especially through living polymerization strategies allowing well-defined blocks and terminal labels (e.g., dyes, contrast agents, charged moieties). Hence, the two elements, peptide and polymer, cooperate to yield materials with unique function and properties quite apart from each alone. Herein, we describe the development of synthetic strategies for accessing these classes of biomolecule polymer conjugates. We discuss the utility of poly(peptide)-based materials in a range of biomedical applications, including imaging of diseased tissues (myocardial infarction and cancer), delivering small molecule drugs to tumors with high specificity, imparting cell permeability to otherwise impermeable peptides, protecting bioactive peptides from proteolysis in harsh conditions (e.g., stomach acid and whole blood), and transporting proteins into traditionally difficult-to-transfect cell types, including stem cells. Poly(peptide) materials offer new properties to both the constituent peptides and to the polymers, which can be tuned by the design of the oligopeptide sequence, degree of polymerization, peptide arrangement on the polymer backbone, and polymer backbone chemistry. These properties establish this approach as valuable for the development of peptides as medicines and materials in a range of settings.
Nature employs a variety of tactics to precisely time and execute the processes and mechanics of life, relying on sequential sense and response cascades to transduce signaling events over multiple ...length and time scales. Many of these tactics, such as the activation of a zymogen, involve the direct manipulation of a material by a stimulus. Similarly, effective therapeutics and diagnostics require the selective and efficient homing of material to specific tissues and biomolecular targets with appropriate temporal resolution. These systems must also avoid undesirable or toxic side effects and evade unwanted removal by endogenous clearing mechanisms. Nanoscale delivery vehicles have been developed to package materials with the hope of delivering them to select locations with rates of accumulation and clearance governed by an interplay between the carrier and its cargo. Many modern approaches to drug delivery have taken inspiration from natural activatable materials like zymogens, membrane proteins, and metabolites, whereby stimuli initiate transformations that are required for cargo release, prodrug activation, or selective transport. This Perspective describes key advances in the field of stimuli-responsive nanomaterials while highlighting some of the many challenges faced and opportunities for development. Major hurdles include the increasing need for powerful new tools and strategies for characterizing the dynamics, morphology, and behavior of advanced delivery systems in situ and the perennial problem of identifying truly specific and useful physical or molecular biomarkers that allow a material to autonomously distinguish diseased from normal tissue.
Highly heterogenous cancers, such as triple-negative breast cancer (TNBC), remain challenging immunotherapeutic targets. Herein, we describe the synthesis and evaluation of immunotherapeutic ...liposomal spherical nucleic acids (SNAs) for TNBC therapy. The SNAs comprise immunostimulatory oligonucleotides (CpG-1826) as adjuvants and encapsulate lysates derived from TNBC cell lines as antigens. The resulting nanostructures (Lys-SNAs) enhance the codelivery of adjuvant and antigen to immune cells when compared to simple mixtures of lysates with linear oligonucleotides both in vitro and in vivo, and reduce tumor growth relative to simple mixtures of lysate and CpG-1826 (Lys-Mix) in both Py230 and Py8119 orthotopic syngeneic mouse models of TNBC. Furthermore, oxidizing TNBC cells prior to lysis and incorporation into SNAs (OxLys-SNAs) significantly increases the activation of dendritic cells relative to their nonoxidized counterparts. When administered peritumorally in vivo in the EMT6 mouse mammary carcinoma model, OxLys-SNAs significantly increase the population of cytotoxic CD8+ T cells and simultaneously decrease the population of myeloid derived suppressor cells (MDSCs) within the tumor micro-environment, when compared with Lys-SNAs and simple mixtures of oxidized lysates with CpG-1826. Importantly, animals administered OxLys-SNAs exhibit significant antitumor activity and prolonged survival relative to all other treatment groups, and resist tumor rechallenge. Together, these results show that the way lysates are processed and packaged has a profound impact on their immunogenicity and therapeutic efficacy. Moreover, this work points toward the potential of oxidized tumor cell lysate-loaded SNAs as a potent class of immunotherapeutics for cancers lacking common therapeutic targets.
Maximizing the tissue-targeting efficiency of nanomaterials while also protecting them from rapid clearance from the bloodstream and limiting their immunogenicity remains a central problem in the ...field of systemic-administered nanomedicine. Herein, we introduce a generalizable strategy to simultaneously increase tumor accumulation, prolong blood circulation, and limit nonspecific immune activation of nanomaterials via peptide-based, tumor-responsive, “sheddable” coatings. Spherical nucleic acids (SNAs) were designed and synthesized to contain an exterior coating composed of zwitterionic polypeptides with recognition sequences for tumor-associated proteases. In the presence of matrix metalloproteinases (MMPs), the polypetide coating is rapidly cleaved, leading to increased cellular uptake of these SNAs, relative to SNAs containing nonsheddable shells. Moreover, the zwitterionic nature of the polypeptide shell shields the SNAs from immune system recognition, which extends their blood circulation time and improves tumor accumulation and in vivo cellular uptake relative to control SNAs with no protective coating. Taken together, these results indicate that this strategy is a viable method for increasing nanoparticle tumor accumulation and can have utility for the systemic delivery of oligonucleotides and nanomaterials to target cells in vivo with low immunogenicity.
Aptamers are nucleic acid-based ligands that exhibit promising features including specific and reversible target binding and inhibition. Aptamers can function as anticoagulants if they are directed ...against enzymes of the coagulation cascade. However, they typically suffer from nucleolytic digestion and fast clearance from the bloodstream. We present thrombin-binding aptamer amphiphiles that self-assemble into nanoscale polymeric micelles with a densely functionalized aptamer-displaying corona. We show that these micellar aptamers retain their native secondary structure in a crowded environment and are stabilized against degradation by nucleases in human serum. Moreover, they are effective inhibitors of human plasma clotting in vitro. The inhibitory effect can be rapidly reversed by complementary nucleic acids that break the aptamers’ secondary structure upon hybridization. Compared to free aptamers, the increased molecular weight and size of the overall assembly promotes extended blood circulation times in vivo.
Access to the brain is restricted by the low permeability of the blood-brain barrier (BBB), greatly hampering modern drug delivery efforts. A promising approach to overcome this boundary is to ...utilize biomacromolecules (peptides, nucleic acids, carbohydrates) as targeting ligands on nanoscale delivery vehicles to shuttle cargo across the BBB. In this mini-review, we highlight the most recent approaches for crossing the BBB using synthetic nanoscale constructs decorated with members of these general classes of biomacromolecules to safely and selectively deliver therapeutic materials to the brain.
This mini-review highlights the most recent advances in the design and application of synthetic nanoscale constructs that utilize biomacromolecular ligands (peptides, nucleic acids, carbohydrates) to target and cross the blood-brain barrier (BBB).
Toll-like receptors (TLRs) are a family of proteins that modulate the innate immune system and control the initiation of downstream immune responses. Spherical nucleic acids (SNAs) designed to ...stimulate single members of the TLR family (e.g., TLR7 or TLR9) have shown utility in cancer immunotherapy. We hypothesized that SNAs synthesized with multiple TLR agonists would enable the simultaneous activation of multiple TLR pathways for maximally synergistic immune activation. Here, we describe the synthesis of SNAs that incorporate both a TLR3 agonist (polyinosinic:polycytidylic acid, poly(I:C)) and TLR9 agonist (CpG oligonucleotide) on the same liposomal scaffold. In this design, CpG comprises the SNA oligonucleotide shell, and poly(I:C) is encapsulated in the liposome core. These dual-TLR activating SNAs efficiently codeliver high quantities of both agonists to the same target cell, yielding enhanced immunostimulation in various murine and human antigen-presenting cells (APCs). Moreover, codelivery of TLR agonists using the SNA both synchronizes and prolongs the duration of costimulatory molecule and major histocompatibility complex class II expression in APCs, which has been shown to be important for efficient downstream immune responses. Taken together, this SNA design provides a strategy for potently activating immune cells and increasing the efficiency of their activation, which likely will inform the preparation of nanomaterials for highly potent immunotherapies.
Liposomal spherical nucleic acids (LSNAs) modified with polyethylene glycol (PEG) units are studied in an attempt to understand how the circulation time and biodistribution of the constructs can be ...manipulated. Specifically, the effect of (1) PEG molecular weight, (2) PEG shell stability, and (3) PEG modification method (PEG in both the core and shell versus PEG in the shell only) on LSNA blood circulation, biodistribution, and in vivo cell internalization in a syngeneic, orthotopic triple-negative breast cancer mouse model is studied. Generally, high PEG molecular weight extends blood circulation lifetime, and a more lipophilic anchor stabilizes the PEG shell and improves circulation and tumor accumulation but at the cost of cell uptake efficiency. The PEGylation strategy has a minor effect on in vitro properties of LSNAs but significantly alters in vivo cell uptake. For example, surface-only PEG in one design contributed to higher in vivo cell internalization than its counterpart with PEG both in the shell and core. Taken together, this work provides guidelines for designing LSNAs that exhibit maximal in vivo cancer cell uptake characteristics in the context of a breast cancer model.
The biological properties of spherical nucleic acids (SNAs) are largely independent of nanoparticle core identity but significantly affected by oligonucleotide surface density. Additionally, the ...payload‐to‐carrier (i.e., DNA‐to‐nanoparticle) mass ratio of SNAs is inversely proportional to core size. While SNAs with many core types and sizes have been developed, all in vivo analyses of SNA behavior have been limited to cores >10 nm in diameter. However, “ultrasmall” nanoparticle constructs (<10 nm diameter) can exhibit increased payload‐to‐carrier ratios, reduced liver accumulation, renal clearance, and enhanced tumor infiltration. Therefore, we hypothesized that SNAs with ultrasmall cores exhibit SNA‐like properties, but with in vivo behavior akin to traditional ultrasmall nanoparticles. To investigate, we compared the behavior of SNAs with 1.4‐nm Au102 nanocluster cores (AuNC‐SNAs) and SNAs with 10‐nm gold nanoparticle cores (AuNP‐SNAs). Significantly, AuNC‐SNAs possess SNA‐like properties (e.g., high cellular uptake, low cytotoxicity) but show distinct in vivo behavior. When intravenously injected in mice, AuNC‐SNAs display prolonged blood circulation, lower liver accumulation, and higher tumor accumulation than AuNP‐SNAs. Thus, SNA‐like properties persist at the sub‐10‐nm length scale and oligonucleotide arrangement and surface density are responsible for the biological properties of SNAs. This work has implications for the design of new nanocarriers for therapeutic applications.
The biological behavior of spherical nucleic acids with ultrasmall 1.4‐nm gold nanocluster cores was compared to conventionally sized spherical nucleic acids with 10‐nm gold nanoparticle cores. Ultrasmall spherical nucleic acids have a higher payload‐to‐carrier ratio and more efficient cellular uptake than larger spherical nucleic acids, as well as prolonged blood circulation, lower liver accumulation, and higher tumor accumulation in vivo.
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