Nanoparticle-hydrogel composite for nucleic acid molecule delivery

Abstract

This disclosure provides novel peptide hydrogels containing encapsulated nanoparticles comprising nucleic acid molecules (such as miRNA) that can undergo multiple gel-to-solution (gel-sol) and solution-to-gel (sol-gel) phase transitions, and their use, such as for controlled delivery of nucleic acid molecules to a subject. Also provided are novel peptides for use in disclosed peptide hydrogels.

Description

[0001] NANOPARTICLE-HYDROGEL COMPOSITE FOR NUCLEIC ACID MOLECULE DELIVERY

[0002] FIELD

[0003] This relates to peptide hydrogels containing encapsulated nanoparticles comprising nucleic acid molecules (such as microRNA), that can undergo multiple gel-to-solution (gel-sol) and solution-to-gel (sol-gel) phase transitions, and their use, for example, for controlled deliver}' of the nucleic acid molecules to a subject.

[0004] ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

[0005] This invention was made with Government Support under project numbers ZIA BC 011657 and ZIA BC 011313 awarded by the National Institutes of Health, National Cancer Institute. The Government has certain rights in this invention.

[0006] SEQUENCE LISTING

[0007] The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and single letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The Sequence Listing is submitted as an XML file in the form of the file named “4239_111365_01_Sequence_Listing” (351,424 bytes), which was created on January 12, 2024, which is incorporated by reference herein.

[0008] BACKGROUND

[0009] The use of injectable hydrogels allows the local delivery of encapsulated therapeutics directly to tissue, limiting systemic distribution of the therapeutic and any associated off-target toxicity. However, current strategies for hydrogel-mediated therapeutic delivery still have challenges that must be overcome before becoming clinically viable, particularly in the context of delivery vehicles for nucleic acid-based therapeutics, such as RNA therapeutics.

[0010] SUMMARY

[0011] Provided herein are implementations of a peptide hydrogel containing encapsulated nanoparticles of a peptide complexed with a nucleic acid molecule (nanoparticle-hydrogel composite). The nanoparticle-hydrogel composite displays shear-thin / recovery mechanical properties, which allow the nanoparticle-hydrogel composite and any additional therapeutic dispersed within the hydrogel to be delivered locally to a target location in a subject, for example, via syringe injection or spray delivery to coat anatomic surfaces at the target location. After delivery, the nanoparticles time-release from the hydrogel matrix to adjacent tissues and are taken up by cells. Once internalized by cells, the nucleic acid molecule is released from the nanoparticle and (depending on the nucleic acid molecule) may affect cellular function.

[0012] The nanoparticles comprise a nucleic acid molecule complexed with a first amphiphilic cationic peptide that is not folded into a p-hairpin conformation. The peptide hydrogel is formed from a fibrillar network of a second amphiphilic cationic peptide that is in a P-hairpin conformation.

[0013] In several aspects, the first amphiphilic cationic peptide comprises or consists of an amino acid sequence set forth as : KKKKKKKKS GGVKVKVKVKVDPPTKVKVKVKV (PKM 1 , SEQ ID NO: 1), wherein theDP is a proline that is a D amino acid, the C-terminus of the peptide is amidated or free carboxylic acid, the N-terminus of the peptide is acetylated or free amine; and the peptide is no more than 50 amino acids in length.

[0014] In several aspects, the second amphiphilic cationic peptide comprises or consists of an amino acid sequence set forth as: VLTKVKTKVDPPTKVEVKVLV (HLT2, SEQ ID NO: 2), wherein theDP is a proline that is a D amino acid, the C-terminus of the second peptide is amidated or free carboxylic acid, the N-terminus of the second peptide is acetylated or free amine, and the second peptide is no more than 50 amino acids in length.

[0015] In some aspects, the nucleic acid molecule complexed with the first amphiphilic cationic peptide is an antisense nucleic acid molecule. In some aspects, the nucleic acid molecule complexed with the first amphiphilic cationic peptide is a miRNA or a mimic and / or mimetic thereof. In some aspects, the nucleic acid molecule complexed with the first amphiphilic cationic peptide is a dinucleotide.

[0016] The peptide hydrogel can further comprise a heterologous anti-cancer agent (such as a chemotherapeutic agent) encapsulated within the peptide hydrogel.

[0017] Also provided are methods of treating or inhibiting a cancer in a subject, comprising administering an effective amount of a disclosed peptide hydrogel to a target location in the subject where the cancer is present or is at risk of being present. In such methods, the nanoparticles in the peptide hydrogel comprise a nucleic acid molecule (such as a miRNA or a mimic and / or mimetic thereof) that inhibits the cancer. The peptide hydrogel comprising the nanoparticles can be administrated to the subject by any suitable means, such as direct injection or spray delivery to the target location in the subject. In some aspects, the nucleic acid molecule complexed with the first amphiphilic cationic peptide is an antisense nucleic acid molecule, the target location is a serosal surface lined by mesothelial cells (for example, part of a serosal body cavity such as the pleural space) in the subject. In some aspects, the cancer is a serosal cancer, such as mesothelioma.

[0018] Also provided is an isolated peptide comprising or consisting of an amino acid sequence set forth as: KKKKKKKKSGGVKVKVKVKVDPPTKVKVKVKV (PKM1, SEQ ID NO: 1), wherein theDP is a proline that is a D amino acid, the C-terminus of the peptide is amidated or free carboxylic acid, the N-terminus of the peptide is acetylated or free amine; and the peptide is no more than 50 amino acids in length. As shown herein, such a peptide unexpectedly good nanoparticle stability, which facilitates production, storage, and anti-cancer efficacy of the peptide hydrogel / nanoparticle composite materials described herein.

[0019] The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

[0020] BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIGs. 1A-1C. Application of surface-fill hydrogel (SFH) to complex surface cancer and mechanism of assembly for its loaded microRNA-peptide polyelectrolyte complex nanoparticles (NPs) by flash nanocomplexation (FNC). (FIG. 1 A) The SFH can be sprayed (or syringe injected) into the pleural cavity following surgical resection of mesothelioma as a treatment adjuvant (or as a primary therapy), where local application can uniformly cover and fill complex tissue surfaces to facilitate delivery of tumor- specific miRNA NPs to remaining tumor cell foci to attenuate their oncogenic signature. (FIG. IB) Highly scalable and kinetically controlled assembly of miRNA- peptide NPs is attainable through FNC, which utilizes a confined impinging jet (CIJ) mixer to achieve turbulent micromixing of the NP components, yielding highly uniform and stable NPs. (FIG. 1C) Assembly of miRNA-peptide polyelectrolyte complex (PEC) NPs occurs over 2 steps. First, miRNA-peptide binding and charge neutralization occurs to produce PEC chains, where secondly, PEC chain condensation and diffusion-limited inter-PEC association leads to the formation of PEC NPs. FNC allows for kinetic control over these processes to aid in isolating these steps, where the characteristic mixing time, TM, of the CIJ mixer (step 1) can occur on a timescale much faster relative to the characteristic assembly time, T , of the PEC NPs (step 2).

[0022] FIGs 2A-2L. FNC production, characterization, and stability of miRNA-MAXl NPs. Effect of input miRNA concentration and characteristic mixing time, TM, on (FIG. 2A) average NP size, Dz, and (FIG. 2B) uniformity by size standard deviation via DLS through varying CIJ mixer inlet flow rate, Q, at a fixed charge ratio (N / P = 10) in RNase-free water. Two regions of mixing kinetics exist, where NPs are most compact and uniform (high Q, TM < TA) and where NPs average larger in size and less uniform (low Q, TM > TA). Data presented as mean ± SD (n = 3). (FIG. 2C) DLS size distributions by intensity for NP preparations (5 pg mL'1miRNA, N / P = 10) by FNC at three different flow rates (Q = 1, 5, and 20 mL min1) representative of mixing kinetics such that TM < TA, TM ~ TA, and TM > TA- Comparing properties of NPs (5 pg mL-1miRNA, N / P = 10) produced by different techniques of FNC (Q = 20 mL min1) and bulk mixing by assessing (FIG. 2D) average NP size, (FIG. 2E) size standard deviation, (FIG. 2F) zeta potential, and (FIG. 2G) miRNA encapsulation. Data presented as mean + SD (ns p > 0.05, unpaired two-tailed t test, n = 3). Batch- to-batch variability evaluated by DLS size distribution by intensity for 3 separate batches produced by (FIG. 2H) FNC or (FIG. 21) bulk mixing. (FIG. 2J) Competition-induced decomplexation of miRNA from NPs (1 pg mL1miRNA, N / P = 10) produced from FNC (Q = 20 mL min ') or bulk mixing by addition of heparin sulfate to compare complex affinity. Data presented as mean + SD (n = 3); dashed lines represent fit to dose-response sigmoidal curve to obtain respective EC50 values. (FIG. 2K) NP colloidal stability assessed over 4 h by monitoring average NP size of NPs (5 pg mL'1miRNA, N / P = 10) formulated by FNC (2 = 20 mL min'1) or bulk mixing. Data presented as mean ± SD (n = 3); dashed line represents fit with simple linear regression (****p < 0.0001 between slopes, ANCOVA). (FIG. 2L) Effect of inlet miRNA concentration and charge ratio on NP colloidal stability assessed over 24 h by monitoring average NP size after formulation by FNC (Q = 20 mL min'1). Data presented as mean ± SD (n = 3).

[0023] FIGs. 3A-3M. FNC production, characterization, and stability of NPs with next-generation cationic peptide designs. (FIG. 3A) Sequences of candidate cationic peptides for miRNA NP formulation, varying in number and position of lysine residues. The sequences shown are the MAXI (VKVKVKVKVDPPTKVKVKVKV, SEQ ID NO: 390), SSP1 (VKVKVDPPTKVKVKVKVKVKV, SEQ ID NO: 391), TSS1 (VKVKVKVKVDPPTKVKVKVDPPKVKVKVKV, SEQ ID NO: 392), 0KM1 (KKKKSGGVKVKVKVKVDPPTKVKVKVKV, SEQ ID NO: 393), and PKM1 (KKKKKKKKSGGVKVKVKVKVDPPTKVKVKVKV, SEQ ID NO: 1) peptides. (FIG. 3B) Binding isotherms for each peptide to scrambled miRNA (0.5 pg mL'1). Lines represent fit to doseresponse sigmoidal curve for EC50 determination; data presented as mean ± SD (ns p < 0.05 for MAXI vs. SSP1, **p < 0.01 for TSSl vs. 0KM1, ****p < 0.0001 otherwise for EC50 comparisons, extra sum-of-squares F test, n = 4). Determination of minimum charge ratio necessary for complete miRNA encapsulation (2 pg mL'1) by (FIG. 3C) MAXI, (FIG. 3D) SSP1, (FIG. 3E) TSS1, (FIG. 3F) 0KM1, and (FIG. 3G) PKM1. Data presented as mean ± SD (n = 3). Effect of characteristic mixing time, M, on (FIG. 3H) average miRNA-PKMl NP size, Dz, and (FIG. 31) uniformity by size standard deviation and scattering signal-to-noise ratio by correlation function measured intercept via DLS through varying CIJ mixer inlet flow rate, Q, at a fixed charge ratio (N / P = 3). Two regions exist of mixing kinetics exist, centered around a TM, optimal, where below (high Q) the mixing is too rapid to proceed past the PEC chain formation step during assembly pathway to NPs, and above (low Q, TM > T ) where NPs are formed but are larger and less uniform. Data presented as mean ± SD (n = 3). Physical properties of NPs formulated with different peptides by FNC (for PKMI: input miRNA concentration 20 pg mL-1, N / P = 3, Q = 6 mL min1; otherwise, input miRNA concentration 5 pg mL1, N / P = 10, Q = 20 mL min1) to compare (FTG. 3J) average NP size, Dz, and (FIG. 3K) zeta potential. Data presented as mean ± SD (n = 3). (FIG. 3L) Longterm NP colloidal stability assessed over 168 h (7 d) for the different miRNA-peptide NPs formulated by FNC (for PKM1 : input miRNA concentration 20 pg mL1, N / P = 3, Q = 6 mL min1; otherwise, input miRNA concentration 5 pg mL1, N / P = 10, Q = 20 mL min1) by monitoring average NP size over time. Data replotted in (FIG. 3M) to exclude miRNA-MAXl NPs for clarity. Data presented as mean + SD (n = 3).

[0024] FIGs. 4A-4F. In vitro evaluation of cellular transfection efficiency and gene silencing capability of FNC-formulated miRNA-peptide NP designs with mesothelioma cells. (FIG. 4A) Histograms from flow cytometry analysis of transfection efficiency in MB52 cells of FNC- formulated FAM-labeled scrambled miRNA-peptide NPs compared to commercially available transfection reagent Lipofectamine™ RNAiMAX (Lipo) after 2 h exposure to NPs (40 nM FAM- miRNA, N / P = 3 for PKMl-based NPs, N / P = 10 for other peptides). Histograms representative of two independent experiments with n = 4 independent samples. (FIG. 4B) Quantified transfection efficiency of FAM-labeled scrambled miRNA-peptide NPs compared to commercially available reagent, Lipofectamine™ RNAiMAX (Lipo), after 2 h exposure (40 nM FAM-miRNA) to MB52 cells (N / P = 3 for PKMl-based NPs, N / P = 10 for other peptides) determined from flow cytometry analysis. Data presented as mean ± SD (ns p > 0.05, ordinary one-way ANOVA with Tukey’s post- hoc test, n = 4, representative of two independent experiments). (FIG. 4C) Cell viability of MB52 cells after 4 h (40 nM miRNA) exposure to scrambled miRNA- or miRNA-215 -based NPs formulated with each peptide design (N / P = 3 for PKM1, N / P = 10 for other peptides) or commercial reagent Lipofectamine™ RNAiMAX (Lipo) as evaluated by MTT assay 96 h postexposure. Data presented as mean ± SD (across miR-215 Lipo, MAXI and PKM1 groups, ns p > 0.05, ordinary one-way ANOVA with Tukey’s post-hoc test; between scramble and miR-215 groups of each respective peptide, ns p > 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired two-tailed t test with Welch’s correction, n = 4, representative of two independent experiments). In vitro evaluation of gene silencing in MB 52 mesothelioma cells for (FIG. 4D) MAD2L1, (FIG. 4E) CDC7, and (FIG. 4F) LMNB2 genes by miR-215 -peptide NPs (40 nM miRNA, N / P = 3 for PKM1, N / P = 10 for other peptides) compared to commercially available reagent Lipofectamine™ RNAiMAX (Lipo) after 4 h exposure and evaluated 48 h post-exposure by qPCR. Expression levels of genes are shown for miR-215 treatment groups relative to scramble miRNA-based NP treatment groups for each respective peptide (all samples normalized to P-actin expression). Data presented as mean ± SD (** / ? < 0.01, ***p < 0.001, ****p < 0.0001, ns p > 0.05, unpaired two- tailed t test conducted between scrambled miRNA and miR-215 groups for each gene and peptide evaluated, n = 3, representative of two independent

[0025] FIGs. 5A-5H. Formulation and physical characterization of SFH loaded with different NP designs. (FIG. 5A) Schematic illustration detailing the loading of NPs into the SFH, where triggered assembly of a hydrogelating peptide mixed with NPs encapsulates them within a fibrillar hydrogel network. (FIG. 5B) In vitro NP release from SFH comparing NPs prepared from MAXI, TSS1, and PKM1 (0.5 wt% HLT2 gels in IxHBS buffer with 1 pg total / gel of FAM-labeled miRNA) over the course of 1 month into a sink of IxHBS buffer (T = 37°C). Data presented as mean ± SD (ns p > 0.05 at 30 d, one-way ANOVA with Tukey’s post-hoc test, n = 3). Dynamic time-sweep shear-thin / recovery of SFH (1 wt% HLT2 in IxHBS buffer, 10 pg mL’1miRNA) loaded with (FIG. 5C) miRNA-MAXl NPs, (FIG. 5D) miRNA-TSSl NPs, or (FIG. 5E) miRNA- PKM1 NPs by oscillatory shear rheology (0.2% strain, 6 rad s’1angular frequency). Shear-thinning was induced at 60 min by applying 1000% strain for 30 s, and subsequent recovery was monitored for 60 min after decreasing strain to 0.2% (6 rad s’1angular frequency). Data presented as mean ± SD (n = 3). (FIG. 5F) Storage modulus (G’) and (FIG. 5G) shear recovery for each NP-loaded SFH (1 wt%) and empty SFH (CTRL, 1 wt% HLT2 gel) evaluated at t = 120 min of respective dynamic time-sweeps (6 rad s’1, 0.2% strain). Data presented as mean + SD (ns p > 0.05, one-way ANOVA with Tukey’s post-hoc test, n = 3). (FIG. 5H) Flow strain for each NP-loaded SFH (1 wt%) and empty SFH (CTRL, 1 wt% HLT2 gel) evaluated from the crossover point of G’ and G” monitored during respective amplitude sweeps (0.1 to 1000% strain at constant 6 rad s’1). Data presented as mean ± SD (ns p > 0.05, one-way ANOVA with Tukey’s post-hoc test, n = 3).

[0026] FIGs. 6A-6C. Tumor uptake and biodistribution of miRNA-peptide PEC NPs released from applied SFH in vivo. (FIG. 6A) Experiment timeline for evaluation of tumor uptake and biodistribution of Cyanine 3(Cy3)-labeled scrambled miRNA-MAXl or -PKM1 NPs from peritumorally injected SFH (1 wt% HLT2, IxHBS buffer, pH 7.4) with a subcutaneous xenograft NSG mouse model bearing established luciferase-expressing MB52 mesothelioma tumors (3xl06cells / mouse, n = 3 mice / group). (FIG. 6B) Analysis of miRNA biodistribution by IVIS imaging of tumor and vital organs (top to bottom: liver, heart, kidney, spleen, lung, and intestine) collected 4 d post-administration of SFH to monitor tumor luminescence (left) and Cy3 fluorescence (right) corresponding to released NPs from SFH representative of each treatment group (empty SFH control, Cy3-miR-MAXl NPs, and Cy3-miR-PKMl NPs loaded into SFH for 100 pg / mL Cy3-miR concentration in gel, n = 3 mice / group). (FIG. 6C) Representative histofluorescence imaging of tumor tissue cross-sections taken from resected subcutaneous xenograft MB52 tumors 4 d postadministration of each peritumorally injected SFH formulation (empty control, Cy3-miR-MAXl NP-loaded, and Cy3-miR-PKMl NP-loaded, 100 pg / mL Cy3-miR concentration in gel). Local release of NPs to tumor tissues evidenced by intracellular Cy3-miRNA fluorescence in MB52 tumor cross-sections; absence of Cy3 fluorescence signal from tumor cross-sections collected from mice of the empty SFH group suggests no tumor tissue autofluorescence is present. Nuclei are stained with DAPI in blue, Cy3-labeled miRNA signal in red; scale bar represents 40 pm (n = 3 mice / group).

[0027] FIGs. 7A-7H. Antitumor efficacy of miRNA-215-peptide PEC NP-loaded SFH against mesothelioma xenograft model in vivo. (FIG. 7A) Experiment timeline for evaluation of antitumor efficacy of miRNA-215-MAXl and miRNA-215-PKMl NPs released from peritumorally injected SFH (1 wt% HLT2, IxHBS buffer, pH 7.4) with a subcutaneous xenograft NSG mouse model bearing established luciferase-expressing MB52 mesothelioma tumors (3xl06cells / mouse, n = 5 mice / group). (FIG. 7B) Tumor luminescence measured by live IVIS imaging at selected time points post-administration of a single application of each SFH formulation (scr-miR-MAXl, miR-215- MAX1, scr-miR-PKMl, miR-215-PKMl ; scr-miR = scrambled miRNA, d = day). Images representative of 5 independent mice for each group (see Supporting Information). (FIG. 7C) Tumor progression as monitored by region of interest (ROI) radiant flux in the live IVIS images at each time point normalized to day 0 for each treatment. Data presented as mean + SEM (*p < 0.05, **p < 0.01, two-tailed Mann-Whitney test conducted at day 30 between scr-miR and miR-215 groups of each respective peptide, n = 5 mice / group). (FIG. 7D) Subcutaneous MB52 tumor volumes measured at each time point post-administration of a single application of each SFH formulation. Data presented as mean + SEM (**p < 0.01, two-tailed Mann- Whitney test conducted at day 30 between scr-miR and miR-215 groups of each respective peptide, n = 5 mice / group). In vivo gene silencing of miR-215-PKMl NPs by a single application of each PKM1 -based SFH formulation 1 week post-administration. (FIG. 7E) Relative expression level of miRNA-215 in resected tumor tissue post-SFH administration (miRNA levels are normalized to U6 snRNA). Data presented as mean ± SEM (*p < 0.05, two-tailed Mann- Whitney test, n = 4 mice / group). miRNA- 215 target gene expression profiles in resected tumor tissue post-SFH administration for (FIG. 7F) MAD2L1, (FIG. 7G) CDC7, and (FIG. 7H) LMNB2 genes (normalized to 0-actin expression level). Data presented as mean ± SEM (**p < 0.01, ***p < 0.001, two-tailed Mann- Whitney test, n = 4 mice / group).

[0028] FIGs. 8A-8I. Cryoprotection selection and production of freeze-dried miRNA-PKMl NPs for long-term storage stability. Selection of optimal cryoprotectant for lyophilization of miRNA- PKMl NPs (20 pg ml1miRNA, N / P = 3) by evaluation of (FIG. 8A) average NP size, Dz, and (FIG. 8B) polydispersity after storage for 24 h in water at room temperature (RT) or addition of cryoprotectant (sucrose, trehalose, or D-mannitol with % as w / v) and subsequent lyophilization and reconstitution in RNase-free water. Data presented as mean ± SD (n = 3). (FIG. 8C) Reconstitution process of miRNA-PKMl NPs lyophilized with 2.5% w / v D-mannitol as cryoprotectant with RNase-free water. Physical properties of miRNA-PKMl NPs freeze-dried with 2.5% w / v D- mannitol as cryoprotectant evaluated at different time points over 3 months of freezer storage (T = - 80°C) to assess (FIG. 8D) average NP size, Dz, (FIG. 8E) polydispersity, (FIG. 8F) zeta potential, and (FIG. 8G) miRNA encapsulation following reconstitution in RNase-free water. Data presented as mean + SD (n = 3). (FIG. 8H) Relative expression level of miRNA-215 in MB52 cells measured by qPCR after lyophilized miRNA-PKMl NP (2.5% w / v D-mannitol as cryoprotectant) treatment (40 nM miR-215 or scrambled (SCR) miRNA) following 1 month storage at 4°C or -80°C and reconstitution in RNase-free water (freshly prepared NPs as a control). H2052 cells were treated with 4 h exposure to NPs and evaluated 48 h post-exposure by qPCR. Expression levels of miRNA- 215 are shown for miR-215 treatment groups relative to SCR treatment groups for each respective storage condition (miRNA levels are normalized to U6 snRNA). Data presented as mean ± SD (between scramble and miR-215 of each respective storage condition, *p < 0.05, ***p < 0.001, unpaired two-tailed t test; across storage conditions of miR-215 groups, ns p > 0.05,mp < 0.001, one-way ANOVA with Tukey’s post-hoc test; n = 3, representative of two independent experiments). (FIG. 81) Evaluation of gene silencing in MB52 mesothelioma cells (MAD2L1, CDC7, and LMNB2) by lyophilized scrambled (SCR) miRNA-PKMl or miR-215PKMl NPs (40 nM miRNA, N / P = 10) either prepared freshly (Fresh) or after lyophilization (2.5% w / v D-mannitol as cryoprotectant), storage for 1 month at 4°C or -80 °C, and reconstitution in RNase-free water. MB 52 cells were treated with 4 h exposure to NPs and evaluated 48 h post-exposure by qPCR. Expression levels of target genes are shown for miR-215 treatment groups relative to SCR treatment groups for each respective peptide (all samples normalized to f>-aclin expression). Data presented as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired two-tailed t test conducted between SCR and miR-215 groups for each gene and storage condition evaluated, n = 3, representative of two independent experiments). FIGs. 9A-9B. (FIG. 9A) Peptide sequence and analytical RP-HPLC trace of purified MAXI peptide (Vydac C18 column, 0-100% Std B over 100 min at 40°C, flow rate = 1 mL min1, X = 220 nm) and (FIG. 9B) ESI (+) mass spectrum of purified MAXI peptide (calculated molecular weight: 2229.9 g mol1).

[0029] FIGs. 10A-10B. (FIG. 10A) Peptide sequence and analytical RP-HPLC trace of purified SSP1 peptide (Vydac C18 column, 0-100% Std B over 100 min at 40°C, flow rate = 1 mL min1, X = 220 nm) and (FIG. 10B) ESI (+) mass spectrum of purified SSP1 peptide (calculated molecular weight: 2229.9 g mol1).

[0030] FIGs. 11A-11B. (FIG. 11 A) Peptide sequence and analytical RP-HPLC trace of purified SSP1 peptide (Vydac C18 column, 0-100% Std B over 100 min at 40°C, flow rate = 1 mL min1, X = 220 nm) and (FIG. 1 IB) ESI (+) mass spectrum of purified SSP1 peptide (calculated molecular weight: 3234.3 g mol’1).

[0031] FIGs. 12A-12B. (FIG. 12A) Peptide sequence and analytical RP-HPLC trace of purified SSP1 peptide (Vydac C18 column, 0-100% Std B over 100 min at 40°C, flow rate = 1 mL min1, X = 220 nm) and (FIG. 12B) ESI (+) mass spectrum of purified OKM1 peptide (calculated molecular weight: 2943.9 g mol1).

[0032] FIGs. 13A-13B. (FIG. 13 A) Peptide sequence and analytical RP-HPLC trace of purified SSP1 peptide (Vydac C18 column, 0-100% Std B over 100 min at 40°C, flow rate = 1 mL min1, X = 220 nm) and (FIG. 13B) ESI (+) mass spectrum of purified PKM1 peptide (calculated molecular weight: 3456.6 g mol1).

[0033] FIGs. 14A-14B. (FIG. 14A) Peptide sequence and analytical RP-HPLC trace of purified SSP1 peptide (Vydac C18 column, 0-100% Std B over 100 min at 40°C, flow rate = 1 mL min1, X = 220 nm) and (FIG. 14B) ESI (+) mass spectrum of purified HLT2 peptide (calculated molecular weight: 3456.6 g mol1).

[0034] FIG. 15. Effect of input miRNA concentration and CIJ mixer inlet jet Reynolds number, Re, on average NP size, DZ, of miRNA-MAXl NPs (fixed N / P = 10) formulated by FNC. Re is dictated by inlet jet flow rate, Q, into the CIJ mixing chamber and the dimensions of the inlet streams. At high Re, turbulent micromixing is likely achieved, yielding shorter characteristic mixing times to allow for homogenous mixing of miRNA and peptide that is not limited by mixing and mass transfer in their respective bulk fluids in the process of complexation. This yields highly uniform NPs at Re > 500 (for highest miRNA concentration analyzed). Dashed line represents fit of data to an exponential decay model; data presented as mean ± SD (n = 3).

[0035] FIGs. 16A-16B. Effect of input miRNA concentration on (FIG. 16A) average NP size (dashed line represents fit with simple linear regression) and (FIG. 16B) zeta potential (dashed line represents average value across all tested formulations) by FNC (Q = 20 mL min-1) and bulk mixing preparation techniques (fixed N / P = 10). Data presented as mean ± SD (n = 3).

[0036] FIGs. 17A-17B. Effect of charge ratio on (FIG. 17A) average NP size (dashed line represents fit with simple linear regression) and (FIG. 17B) zeta potential (dashed line represents average value across all tested formulations) by FNC (Q = 20 mL min-1) and bulk mixing preparation techniques (fixed inlet miRNA concentration 2 pg mL-1). Data presented as mean ± SD (n = 3).

[0037] FIG. 18. Fluorescence signal of individual NP components after addition of RiboGreen assay reagent (scramble miRNA concentration = 0.5 pg mL-1, peptide concentrations = 25 pM), showing no reaction between peptides and RiboGreen reagent. Data presented as mean ± SD (n = 4).

[0038] FIGs. 19A-19G. Circular dichroism (CD) spectra of peptides alone and in complex with scrambled miRNA in RNase-free water (peptide concentration = 35 pM, charge ratio N / P = 10, T = 25°C). (FIG. 19A) CD spectra of all peptides alone in water, each adopting intrinsically disordered state (random coil). (FIG. 19B) CD spectra of all miRNA-peptide NPs in water, showing peptides largely maintain disordered state after complexation to miRNA. Direct comparisons of free peptide to their respective miRNA complexes for (FIG. 19C) MAXI, (FIG. 19D) SSP1, (FIG. 19E) TSS1, (FIG. 19F) 0KM1, and (FIG. 19G) PKM1. Data presented as mean of n = 5 scans.

[0039] FIGs. 20A-20B. Competition-induced decomplexation of miRNA from NPs (10 pg mL-1 miRNA, N / P = 10) by addition of heparin sulfate to compare complex affinity. (FIG. 20A) Doseresponse of miRNA decomplexation by increasing heparin sulfate concentration; lines represent fit of data to dose-response sigmoidal curve to obtain respective EC50 values. Data presented as mean + SD (n - 3). (FIG. 20B) Table of EC50 values obtained from curve fits; pairwise comparisons evaluated with extra sum-of- squares F test (ns p > 0.05, *p < 0.05, **p < 0.01, ****p < 0.0001).

[0040] FIGs. 21A-21B. Effect of characteristic mixing time, rM, on (FIG. 21 A) average miRNA- TSS 1 NP size, DZ, and (FIG. 21B) uniformity by size standard deviation via DLS through varying CIJ mixer inlet flow rate, Q, in RNase-free water (inlet miRNA concentration = 5 pg mL-1, N / P = 10). Two regions of mixing kinetics exist, where NPs are most compact and uniform (high Q, rM < rA) and where NPs average larger in size and are less uniform (low Q, rM > rA). Data presented as mean ± SD (n = 3).

[0041] FIGs. 22A-22D. Exploring factors impacting the assembly of miRNA-PKMl polyelectrolyte complexes by both FNC and bulk mixing. (FIG. 22A) Monitoring DLS correlation function measured intercept in miRNA-PKMl complexation and assembly to NP with increasing input concentrations of miRNA (at fixed N / P = 10) with bulk mixing. Dashed line represents fit of data to a dose-response sigmoidal curve to obtain EC50 value (EC50 = 19.8 pg mL-1 miRNA); data presented as mean ± SD (n = 3). (FIG. 22B) Representative correlation functions of miRNA- PKM1 complexes (input miRNA concentration = 25 pg mL-1, N / P = 10) formed by FNC (Q = 20 mL min-1) or bulk mixing. Experiments were repeated for 3 batches with similar results. (FIG. 22C) Representative correlation functions of miRNA-PKMl complexes (input miRNA concentration = 50 pg mL-1, N / P = 10) formed by FNC (Q = 20 mL min-1) or bulk mixing.

[0042] Experiments were repeated for 3 batches with similar results. (FIG. 22D) Representative correlation functions of miRNA-PKMl complexes (input miRNA concentration = 10 pg mL-1 , N / P = 3) formed by FNC (Q = 20 mL min-1) or bulk mixing. Experiments were repeated for 3 batches with similar results.

[0043] FIGs. 23A-23B. Competition-induced decomplexation of miRNA from miRNA-PKMl polyelectrolyte complexes (10 pg mL-1 miRNA) by addition of heparin sulfate to compare complex affinity at different charge ratios (N / P = 3 or 10). (FIG. 23A) Dose-response of miRNA decomplexation by increasing heparin sulfate concentration; lines represent fit of data to doseresponse sigmoidal curve to obtain respective EC50 values. Data presented as mean± SD (n = 3). (FIG. 23B) Table of EC50 values obtained from curve fits; pairwise comparisons evaluated with extra sum-of-squares F test (****p < 0.0001).

[0044] FIG. 24. Representative DLS correlation functions and size distributions by intensity for miRNA-PKMl NP preparations by FNC (input miRNA concentration = 20 pg mL-1, N / P = 3) at three different flow rates (Q = 1, 6, and 15 mL min-1) representative of mixing kinetics such that rM < rM, optimal, rM ~ rM, optimal, and rM > rM, optimal. Data are representative of n = 3 batches for each condition.

[0045] FIG. 25. Batch-to-batch variability evaluated by DLS size distribution by intensity for 3 separate batches of miRNA-PKMl NPs produced by FNC (Q = 6 mL min-1, inlet miRNA concentration = 20 pg mL-1, N / P = 3).

[0046] FIGs. 26A-26B. (FIG. 26A) Representative transmission electron microscopy (TEM) image of miRNA-PKMl NPs formulated by FNC (miRNA concentration = 20 pg mL-1, N / P = 3, Q = 6 mL min-1). Scale bar represents 100 nm. (FIG. 26B) Population frequency distribution for observed miRNA-PKMl NP diameters analyzed by TEM (15 bins, 10 nm each), where p represents average particle diameter given as mean ± SD alongside median (M) measured diameter (n = 205 analyzed particles). Dotted line represents fit of the data to a Gaussian distribution.

[0047] FIG. 27. Long-term NP colloidal stability assessed over 3 months (at RT in RNase-free water) for miRNA-PKMl NPs formulated by FNC (input miRNA concentration 20 pg mL-1, N / P = 3, Q = 6 mL min-1) by monitoring average NP size over rime by DLS. Data presented as mean ± SD (n = 3).

[0048] FIG. 28. Cell viability of MB52 cells after 4 h (40 nM miRNA) exposure to scrambled miRNA- or miRNA-215-based NPs formulated with each peptide design (N / P = 3 for PKM1, N / P = 10 for other peptides) or commercial reagent Lipofectamine RNAiMAX (Lipo) as evaluated by MTT assay 96 h post-exposure. Data presented as mean ± SD (across miR-215-peptide groups, #p < 0.05, ##p < 0.01, ns p > 0.05, ####p < 0.0001 otherwise, ordinary one-way ANOVA with Tukey’s post-hoc test; between scramble and miR-215 groups of each respective peptide, ns p > 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired two-tailed t test with Welch’s correction; n = 4, representative of two independent experiments).

[0049] FIGs. 29A-29B. In vitro evaluation of cellular transfection efficiency and cytotoxicity of miRNA-peptide NPs with H2052 mesothelioma cells. (FIG. 29A) Transfection efficiency of FAM- labeled scrambled miRNA-peptide NPs compared to commercially available reagent, Lipofectamine™ RNAiMAX (Lipo), after 2 h exposure (40 nM FAM-miRNA) to H2052 cells (N / P = 3 for PKM1 -based NPs, N / P = 10 for other peptides) determined from flow cytometry analysis. Data presented as mean ± SD (*p < 0.05, ***p < 0.001, ns p > 0.05 otherwise, ordinary one-way ANOVA with Tukey’s post-hoc test, n = 4, representative of two independent experiments). (FIG. 29B) Cell viability of H2052 cells after 4 h (40 nM miRNA) exposure to scrambled miRNA- or miRNA-215-based NPs formulated with each peptide design (N / P = 3 for PKM1, N / P = 10 for other peptides) or commercial reagent Lipofectamine™ RNAiMAX (Lipo) as evaluated by MTT assay 96 h post-exposure. Data presented as mean ± SD (across miR-215-peptide groups, #p < 0.05, ##p < 0.01, ns p > 0.05, ####p < 0.0001 otherwise, ordinary one-way ANOVA with Tukey’s post-hoc test; between scramble and miR-215 groups of each respective peptide, *p < 0.05, **p < 0.01, ****p < 0.0001, unpaired two-tailed t test with Welch’s correction; n = 4, representative of two independent experiments).

[0050] FIG. 30. In vitro evaluation of gene silencing of oncogenesis in MB52 mesothelioma cells (MAD2L1, CDC7, and LMNB2) by scrambled (SCR) miRNA-peptide or miR-215-peptide NPs (40 nM miRNA, N / P = 3 for PKM1, N / P = 10 for other peptides) compared to commercially available reagent Lipofectamine (Lipo) after 4 h exposure and evaluated 48 h post-exposure by qPCR. Expression levels of miRNA are shown for miR-215 treatment groups relative to SCR treatment groups for each respective peptide (all samples normalized to P-actin expression). Data presented as mean ± SD (n = 3, representative of two independent experiments).

[0051] FIG. 31. In vitro NP release from SFH loaded with NPs prepared from MAXI and PKM1 (1 wt% HLT2 gels in IxHBS buffer with 1 pg total / gel of FAM-labeled miRNA) over the course of 2 weeks into a sink of IxHBS buffer (T = 37°C). Data presented as mean ± SD (ns p > 0.05 at 14 d, two-tailed unpaired t test, n = 3).

[0052] FIG. 32. Dynamic time-sweep shear-thin / recovery of empty SFH (1 wt% HLT2 in IxHBS buffer) by oscillatory rheology (0.2% strain, 6 rad s-1 angular frequency). Shear-thinning was induced at 60 min by applying 1000% strain for 30 s, and subsequent recovery was monitored for 60 min by decreasing strain to 0.2% (6 rad s-1 angular frequency). Data presented as mean ± SD (n = 3).

[0053] FIGs. 33A-33H. Frequency and amplitude sweeps by oscillatory shear rheology of miRNA-peptide NP-loaded SFH (1 wt% HLT2 in IxHBS buffer, 10 pg mL-1 miRNA) monitoring storage (G’) and loss (G”) modulus. Frequency sweeps conducted at constant 0.2% strain and increasing angular frequency (0.1 to 100 rad s-1) for (FIG. 33A) empty SFH or SFH loaded with (FIG. 33B) miRNA-MAXl NPs, (FIG. 33C) miRNA-TSSl NPs, or (FIG. 33D) miRNA-PKMl NPs. Data presented as mean ± SD (n = 3). Amplitude sweeps conducted at constant angular frequency of 6 rad s-1 and increasing strain (0.1 to 1000% strain) for (FIG. 33E) empty SFH or SFH loaded with (FIG. 33F) miRNA-MAXl NPs, (FIG. 33G) miRNA-TSSl NPs, or (FIG. 33H) miRNA-PKMl NPs. Data presented as mean ± SD (n = 3).

[0054] FIGs 34A and 34B. Particle size and size distributions for scramble miRNA-PKMl NP preparations by FNC for in vivo studies (input miRNA concentration = 400 pg mL’1, N / P = , Q = 6 mL min1) (FIG. 34 A) In vzvo-scale PKM1 formulations exhibit colloidal stability over at least 7 d, as assessed by monitoring average NP size by DLS. (FIG. 34B) Representative size distribution (by intensity) of miR-PKMl NPs formulated at in vivo scale by DLS. Data presented as mean ± SD (n = 3 batches each).

[0055] FIGs. 35A-35B. Monitoring Cy3 -labeled scrambled miRNA-MAXl or -PKM1 NP release and biodistribution from peritumorally-injected SFH (1 wt% HLT2, IxHBS buffer, pH 7.4) in a subcutaneous xenograft NSG mouse model with luciferase-expressing MB52 mesothelioma cells. Tumor luminescence (left) and Cy3 fluorescence (right) measured by IVIS imaging on (FIG. 3 A) day 0 after injection (dO) and (FIG. 35B) day 4 (d4) for each treatment group (empty SFH control, Cy3-miR-MAXl NPs and Cy3-miR-PKMl NPs loaded into SFH for 100 pg / mL Cy3-miR concentration in gel, n = 3 mice / group).

[0056] FIG. 36. Tumor luminescence monitored over 30 days after a single SFH application in a subcutaneous xenograft NSG mouse model with luciferase-expressing MB52 mesothelioma cells via IVIS imaging. Treatment groups of SFH (1 wt% HLT2, IxHBS buffer, pH 7.4) were loaded with either scrambled miRNA-based (scr-miR) NPs formulated with MAXI or PKM1 (100 pg / mL miRNA concentration in gel) or miRNA-215 (miR-215) NPs formulated with MAXI or PKM1 (100 pg / mL miRNA total in gel). Imaging conducted on day 1, 9, 24, and 30 after SFH application (d = day) with n = 5 mice / group.

[0057] FIGs. 37A-37D. Evaluating the efficacy of gene silencing in vivo of miRNA-215 target genes by a single application of SFH (1 wt% HLT2, IxHBS buffer, pH 7.4, loaded with scrambled miRNA- (scr-miR-MAXl) or miRNA-215-based (miR-215-MAXl) MAXI NPs, 100 pg / mL miRNA concentration in gel) in a subcutaneous xenograft NSG mouse model with luciferaseexpressing MB52 mesothelioma cells 1 week after application. (FIG. 37A) Relative expression level of miRNA-215 in resected tumor tissue post-SFH administration (miRNA levels are normalized to U6 snRNA). Data presented as mean ± SEM (*p < 0.05, two-tailed Mann- Whitney test, n - 4 mice / group). Target gene expression profiles in resected tumor tissue post-SFH administration for (FIG. 37B) MAD2L1, (FIG. 37C) CDC7, and (FIG. 37D) LMNB2 genes (normalized to (Lactin expression level). Data presented as mean ± SEM (**p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired Mann- Whitney test, n = 4 mice / group).

[0058] FIGs. 38A-38D. Particle size and size distributions by intensity for scramble miRNA- MAX1 and scramble miRNA-PKMl NP preparations by FNC for in vivo studies (input miRNA concentration = 400 pg mL-1, N / P = 10 for MAXI, N / P = 3 for PKM1) at their respective flow rates (Q = 20 mL min-1 for MAXI and Q = 6 mL min-1 for PKM1). (FIG. 38A) Average NP size by DLS comparing scr-miR-MAXl and scr-miR-PKMl formulations. (FIG. 38B) In wm-scale PKM1 formulations exhibit colloidal stability over at least 7 d, as assessed by monitoring average NP size by DLS. Representative size distributions by intensity of (FIG. 38C) miR-MAXl and (FIG. 38D) miR-PKMl NPs formulated at in vivo scale by DLS. Data presented as mean ± SD (n = 3 batches each).

[0059] FIGs. 39A-39C. Dynamic time-sweep shear-thin / recovery of SFH (1 wt% HLT2 in IxHBS buffer, 10 pg mL-1 miRNA) loaded with (FIG. 39A) lyophilized miRNA-MAXl NPs or (FIG. 39B) lyophilized miRNA-PKMl NPs (2.5% w / v D-mannitol as cryoprotectant) by oscillatory shear rheology (0.2%strain, 6 rad s-1 angular frequency). Shear-thinning was induced at 60 min by applying 1000% strain for 30 s, and subsequent recovery was monitored for 60 min after decreasing strain to 0.2% (6 rad s-1 angular frequency). Data presented as mean ± SD (n = 3). (FIG. 39C) Storage modulus (G’) for each lyophilized NP-loaded SFH (1 wt%) and fresh NP-loaded SFH (1 wt%, data reproduced from Figure 5F) evaluated at t = 120 min of respective dynamic time-sweeps (6 rad s-1, 0.2% strain). Data presented as mean ± SD (*p < 0.05, ns p > 0.05, one-way ANOVA with Tukey’s post-hoc test, n - 3).

[0060] FIGs. 40A-40D. Frequency and amplitude sweeps by oscillatory shear rheology of lyophilized miRNA-peptide NP-loaded SFH (1 wt% HLT2 in IxHBS buffer, 10 pg mL-1 miRNA, 2.5% w / v D-mannitol as cryoprotectant) monitoring storage (G’) and loss (G”) modulus. Frequency sweeps conducted at constant 0.2% strain and increasing angular frequency (0.1 to 100 rad s-1) for SFH loaded with (FIG. 40A) lyophilized miRNA-MAXl NPs and (FIG. 40B) lyophilized miRNA-PKMl NPs. Data presented as mean ± SD (n = 3). Amplitude sweeps conducted at constant angular frequency of 6 rad s-1 and increasing strain (0.1 to 1000% strain) for SFH loaded with (FIG. 40C) lyophilized miRNA-MAXl NPs and (FIG. 40D) lyophilized miRNA- PKMl NPs. Data presented as mean ± SD (n = 3).

[0061] DETAILED DESCRIPTION

[0062] Administration of heterologous nucleic acid molecules (such as miRNA) can alter cellular activity to provide a therapeutic effect. However, translating miRNA and other nucleic acid molecules into treatments for human disease has been hampered by the lack of clinical delivery vehicles. Described herein are aspects of a novel nanoparticle-hydrogel composite that can deliver nucleic acid molecules (such as miRNA) to target tissue sites and facilitate transport of the nucleic acid molecule into cells. The nanoparticle-hydrogel composite includes nanoparticles comprised of an amphiphilic cationic peptide (e.g., PKM1) complexed to the nucleic acid molecule. The nanoparticles are encapsulated into a shear-thinning peptide-based hydrogel, which is comprised of a fibrillar network of self-assembled cationic amphiphilic peptides (e.g., HLT2).

[0063] Aspects of the disclosed nanoparticle-hydrogel composite display shear-thin / recovery mechanical properties, which allow the nanoparticle-hydrogel composite and any additional therapeutic dispersed within the hydrogel to be delivered locally to a body cavity via percutaneous or surgical access by syringe injection or sprayed to coat anatomic surfaces. For example, the nanoparticle-hydrogel composite can be applied in the space between the parietal and visceral pleura of the lung after surgical removal of mesothelioma tumor. After application, the nanoparticles are time-released from the hydrogel matrix to adjacent tissues and taken up by cells. Once internalized by cells, the nucleic acid molecule is released from the nanoparticle to affect cellular function. In addition to nucleic acid nanoparticles, the hydrogel can carry other biologic agents e.g. , encapsulated chemotherapeutics) permitting combinatorial therapies.

[0064] While many different nanomaterials can package RNA into non-viral vectors by various mechanisms, polyelectrolyte complexation is a highly facile means to form RNA-loaded NPs with high translational potential. Here, supercharged cationic species are introduced to negatively charged RNA, where the interacting molecules complex and condense into NPs after charge neutralization; however, NPs formulated this way face the same challenges as other materials for clinical translation, such as reproducible size and uniformity and aggregation potential. As shown herein, many tested peptides resulted in metastable NPs, where their size distribution begins to broaden over time if not quickly encapsulated into the gel phase, thereby complicating transition to large-scale, clinical settings. However, the newly designed PKM1 peptide is shown to form highly stable nanoparticles with miRNA, which facilitates production, storage, and anti-cancer efficacy of the peptide hydrogel.

[0065] In an exemplified aspect, the nanoparticle-hydrogel composite is used to treat mesothelioma, a complex surface cancer of the lung lining. This recalcitrant tumor is generally resistant to chemotherapy, radiation, and cannot be completely excised. Thus, there is an urgent need for improved mesothelioma treatment strategies. Beyond mesothelioma, the nanoparticlehydrogel composite can be used to deliver nucleic acid molecule (such as miRNA) and / or other combination therapies to diverse types of surface cancers.

[0066] A. Summary of Terms

[0067] Unless otherwise noted, technical terms are used according to conventional usage. As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “a peptide” includes single or plural peptides and can be considered equivalent to the phrase “at least one peptide.” As used herein, the term “comprises” means “includes.” Thus, “comprising a peptide” means “including a peptide” without excluding other elements. It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described below. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided:

[0068] About: Unless context indicated otherwise, “about” refers to plus or minus 5% of a reference value. For example, “about” 100 refers to 95 to 105.

[0069] Amphiphilic cationic 0-hairpin peptide: A peptide that has a positive electrostatic charge at neutral pH and folds into a [3-hairpin conformation under suitable conditions, such as when dissolved at 2.0% w / v in 50 mM Bis Tris Propane, pH 7.4, 150 mM NaCl, at 25°C. When folded into the p-hairpin conformation, one face of the hairpin is primarily hydrophobic, and the other is primarily hydrophilic. A non-limiting example of an amphiphilic cationic P-hairpin peptide is provided herein as HLT2 peptide. Antisense nucleic acid molecule: An oligomeric compound that is at least partially complementary to the region of a target nucleic acid molecule (such as a RNA gene product) to which it hybridizes. As used herein, an antisense compound that is “specific for” a target nucleic acid molecule is one which specifically hybridizes with and modulates expression of the target nucleic acid molecule. As used herein, a “target” nucleic acid is a nucleic acid molecule to which an antisense compound is designed to specifically hybridize and modulate expression.

[0070] Non- limiting examples of antisense nucleic acid molecules include antisense oligonucleotides, siRNAs, miRNAs, shRNAs and ribozymes. As such, these nucleic acid molecules can be introduced as single-stranded, double-stranded, circular, branched or hairpin nucleic acid molecules and can contain structural elements such as internal or terminal bulges or loops. Double-stranded antisense nucleic acid molecules can be two strands hybridized to form double- stranded compounds or a single strand with sufficient self complementarity to allow for hybridization and formation of a fully or partially double-stranded nucleic acid molecule.

[0071] |3-hairpin conformation: A structural conformation of a peptide or protein. The P-hairpin conformation includes two 3-strands linked by a 3-turn to form a “hairpin”-like shape. The structure is amphiphilic; thus, one face of the hairpin is primarily hydrophobic, and the other is primarily hydrophilic. A limited number of the side chains of hydrophobic amino acids can exist on the hydrophilic face of the hairpin and vice versa, but not so many as to change the overall amphiphilicity of the folded structure. A non-limiting example of a peptide that can fold into an 3- hairpin conformation is provided herein as HLT2.

[0072] Cancer: A malignant neoplasm (e.g., a tumor) that has undergone characteristic anaplasia with loss of differentiation, increased rate of growth, invasion of surrounding tissue, and is capable of metastasis. Metastatic cancer is a cancer at one or more sites in the body other than the site of origin of the original (primary) cancer from which the metastatic cancer is derived. In some examples, cancer is a condition in which expression of one or more miRNAs is altered (for example, increased or decreased) in the neoplasm, compared to normal or healthy tissue of the same tissue type.

[0073] Chemotherapeutic agent: Any chemical agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancer. In one aspect, a chemotherapeutic agent is an agent of use in treating a serosal neoplasm, such as mesothelioma. Non-limiting examples of chemotherapeutic agents that can be used include microtubule binding agents, DNA intercalators or cross-linkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. Disperse: Distribute throughout a medium, such as a disclosed peptide hydrogel. In particular examples, nanoparticles composed of a miRNA complexed with an amphiphilic cationic peptide are dispersed in a peptide hydrogel and are distributed evenly throughout the peptide hydrogel. However, dispersal of the nanoparticles in a peptide hydrogel does not require absolute even distribution.

[0074] Effective amount: An amount of an agent (such as one or more miRNAs) that is sufficient to produce a desired response, such as reducing or inhibiting one or more signs or symptoms associated with a condition or disease. In some examples, an “effective amount” is an amount that treats or inhibits one or more signs or symptoms of a tumor. In some examples, an “effective amount” is a therapeutically effective amount in which the agent alone or with one or more additional therapies, induces the desired response, such as a decrease in size of a tumor in a subject, number of tumors in a subject, size or number of tumor metastases in a subject, and / or an increase in survival of a subject (such as disease-free survival, metastasis -free survival, or overall survival).

[0075] Expression vector: A vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis- acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Non-limiting examples of expression vectors include cosmids and DNA plasmids that incorporate the recombinant polynucleotide.

[0076] Isolated: An “isolated” biological component (such as a nucleic acid molecule, protein, or cell) has been substantially separated or purified away from other biological components (for example, in the cell or tissue of an organism, or the organism itself, in which the component naturally occurs, such as other chromosomal and extra-chromosomal DNA and RNA, proteins and cells). Nucleic acid molecules and proteins that have been “isolated” include those purified by standard purification methods. The term also embraces nucleic acid molecules (including miRNAs) and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acid molecules and proteins. microRNA (miRNA or miR): Single-stranded, small non-coding RNA molecules that regulate gene expression. miRNAs are generally about 16-27 nucleotides in length. miRNAs typically modulate gene expression (e.g., increase or decrease translation) by promoting cleavage of target mRNAs or by blocking translation of the cellular transcript. miRNAs are processed from primary transcripts known as pri-miRNA to short stem-loop structures called precursor (pre)- miRNA and finally to functional, mature miRNA. Mature miRNA molecules are partially complementary to one or more messenger RNA molecules, and their primary function is to down- regulate gene expression. As utilized herein, “miR nucleic acid” or “miRNA nucleic acid” refers to any of a pri-miRNA, a pre-miRNA, a miRNA duplex, or a mature miRNA.

[0077] A nomenclature scheme is established for miRNAs. For example, a miRNA name includes a three or four letter species prefix, such as “hsa” for Homo sapiens, and a numeric suffix, such as “1,” resulting in a complete name of “hsa-miR-1.” Mature miRNA sequences expressed from more than one hairpin precursor molecule are distinguished by “-1” and “-2” (such as hsa-miR-24-1 and hsa-miR-24-2). Related hairpin loci expressing related mature miRNA sequences have lettered suffixes (such as hsa-miR-26a and hsa-miR-26b). In some cases, mature miRNAs from both the 5’ and 3’ arms of the hairpin precursor are identified, which are designated “5p” or “3p”, respectively (such as hsa-miR-27b-5p and hsa-miR-27b-3p).

[0078] Most known miRNA sequences are publicly available. For example, miRBase (mirbase.org) includes a searchable database of annotated miRNA sequences. miRNA sequences are also available through other databases known to one of ordinary skill in the art, including the National Center for Biotechnology Information (ncbi.nlm.nih.gov). One of ordinary skill in the art can also identify targets for specific miRNAs utilizing public databases and algorithms, for example at MicroCosm Targets (ebi.ac.uk / enright-srv / microcosm / htdocs / targets / ), TargetScan (targetscan.org), and PicTar (pictar.mdc-berlin.de). Based on miRNA sequences from one organism (such as mouse), one of ordinary skill in the art can utilize the available databases to determine a corresponding miRNA from another organism (such as human). miRNA Mimic or Mimetic: A miRNA mimetic includes a miRNA has the same sequence as the native or wild type miRNA, but has a modified backbone, a modified base, and / or a 5’ or 3’ end modification. In some examples a miRNA mimetic is may less susceptible to degradation or nuclease activity. A miRNA mimic is a miRNA with at least one sequence modification and having 75% or higher sequence identity to a native or wild type miRNA and that also binds to the same mRNA(s) with similar affinity as the wild type or native miRNA. The disclosed miRNAs may also be both a miRNA mimetic and a miRNA mimic, for example, a miRNA with at least one sequence modification (e.g., 75% or higher sequence identity) to a wild type miRNA, and also having a modified backbone, base, and / or end modification.

[0079] Nanoparticles: Solid colloidal particles that range in size from about 10-1000 nm. They can be made from biodegradable and biocompatible biomaterials. Nanoparticles used in the disclosed aspects include a cationic peptide complexed with a nucleic acid molecule via electrostatic interactions that can be taken up into cells in vivo and in vitro.

[0080] Nucleic acid molecule: A deoxyribonucleotide or ribonucleotide polymer in either single or double stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. A nucleic acid molecule includes both sense and anti-sense strands of RNA, mRNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. A nucleic acid molecule is usually at least 10 bases in length, unless otherwise specified. In some examples, a nucleic acid molecule used with the disclosed peptide hydrogel is a dinucleotide.

[0081] Peptide: A chain of amino acids, typically less than 75 amino acids in length, such as 20- 50 amino acids in length. The residues in a peptide can include post-translational or secondary modifications, such as glycosylation, sulfation or phosphorylation, as well as chemical modifications. “Peptide” applies to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers, including amino acid polymers in which one or more amino acid residues are non-natural amino acids. A “residue” refers to an amino acid or amino acid mimetic incorporated in a peptide by an amide bond or amide bond mimetic. A peptide has an amino terminal (N-terminal) end and a carboxy terminal (C-terminal) end.

[0082] Typically, the amino acids making up a peptide are numbered in order, starting at the amino terminus and increasing in the direction toward the carboxy terminus of the peptide. Thus, when one amino acid is said to “follow” another, that amino acid is positioned closer to the carboxy terminal end of the peptide than the preceding amino acid.

[0083] Peptide hydrogel: A colloid gel including an internal phase and a dispersion medium, in which an aqueous solution is the dispersion medium and a self-assembled network of peptides is the internal phase. The peptides in the hydrogel are self-assembled and are folded into an p-hairpin conformation in the fibrillar network that forms the internal phase of the hydrogel. The peptide hydrogels disclosed herein are made using peptides that form an P-hairpin conformation in an aqueous solution comprising 150 mM NaCl and a pH of 7.4 at 25-37 °C. Thus, an aqueous solution containing 2% w / v of a disclosed peptide and 150 mM NaCl and a pH of 7.4 forms a peptide hydrogel comprising a fibrillar network of the peptide when incubated at 25-37 °C in a container. Peptide hydrogels include a sufficient elastic modulus or stiffness that allows them to maintain shape. In several aspects, the peptide hydrogel has an elastic modulus of 40 Pascal or greater. Peptide hydrogels formed from the disclosed self-assembled peptides in an P-hairpin conformation can be characterized by shear- thin / reco very rheological properties. The hydrogel undergoes a gelsol phase transition upon application of shear stress, and a sol-gel phase transition upon removal of the shear stress. Thus, application of shear stress converts the solid-like gel into a viscous gel capable of flow, and cessation of the shear results in gel recovery. General information concerning peptide hydrogels having shear-thin / recovery rheological properties and methods of making same is provided, for example, in Sathaye, et al. Biomacromolecules, 2014, 15(11 ):3891-3900; Huie et al., 2008, Faraday Discuss, 139:251-420. In several aspects, the peptide hydrogel can be a sterile hydrogel prepared with physiological and non-toxic dispersion medium for use to deliver therapeutics to a subject.

[0084] Peptide modifications: The present disclosure includes synthetic peptides, as well as derivatives (chemically functionalized polypeptide molecules obtained starting with the disclosed polypeptide sequences) and variants (homologs) of peptides described herein. The peptides disclosed herein include a sequence of amino acids that can include L- and / or D- amino acids, naturally occurring and otherwise.

[0085] Peptides can be modified by a variety of chemical techniques to produce derivatives having essentially the same activity as the unmodified polypeptides, and optionally having other desirable properties. For example, carboxylic acid groups of the protein, whether carboxyl-terminal or side chain, may be provided in the form of a salt of a pharmaceutically-acceptable cation or esterified to form a C1-C16 ester, or converted to an amide of formula NR1R2 wherein Ri and R are each independently H or C1-C16 alkyl, or combined to form a heterocyclic ring, such as a 5- or 6- membered ring. Amino groups of the polypeptide, whether amino- terminal or side chain, may be in the form of a pharmaceutically-acceptable acid addition salt, such as the HC1, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts, or may be modified to C1-C16 alkyl or dialkyl amino or further converted to an amide.

[0086] Hydroxyl groups of the polypeptide side chains can be converted to C1-C16 alkoxy or to a C1-C16 ester using well-recognized techniques. Phenyl and phenolic rings of the polypeptide side chains can be substituted with one or more halogen atoms, such as F, Cl, Br or I, or with C1-C16 alkyl, Ci-C 16 alkoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids. Methylene groups of the polypeptide side chains can be extended to homologous C2-C4 alkylenes. Thiols can be protected with any one of a number of well -recognized protecting groups, such as acetamide groups. Those skilled in the art will also recognize methods for introducing cyclic structures into the polypeptides of this disclosure to select and provide conformational constraints to the structure that result in enhanced stability. For example, a C- or N-terminal cysteine can be added to the polypeptide, so that when oxidized the polypeptide will contain a disulfide bond, generating a cyclic polypeptide. Other polypeptide cyclizing methods include the formation of thioethers and carboxyl- and amino-terminal amides and esters.

[0087] Subject: Living multi-cellular vertebrate organisms, a category that includes human and non-human mammals. In one example, a subject is a human.

[0088] Treating or preventing a disease: “Preventing” a disease refers to inhibiting the full development of a disease, for example in a person who is known to have a predisposition to a disease such as a cancer. “Treating” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. “Ameliorating” refers to the reduction in the number or severity of signs or symptoms of a disease, such as cancer. In several aspects, treatment refers to a reduction in size of a tumor, a decrease in the number and / or size of metastases, or a decrease in a symptom of the tumor.

[0089] Tumor burden: The total volume, number, metastasis, or combinations thereof of tumor or tumors in a subject, or in an organ of a subject.

[0090] Under conditions sufficient for: A phrase that is used to describe any environment that permits a desired activity. In one example the desired activity is treatment of a tumor.

[0091] B. Isolated Peptides

[0092] Provided wherein is an isolated peptide comprising or consisting of an amino acid sequence set forth as: KKKKKKKKSGGVKVKVKVKVDPPTKVKVKVKV (PKM1, SEQ ID NO: 1), wherein theDP is a proline that is a D amino acid, the C-terminus of the peptide is amidated or free carboxylic acid, the N-terminus of the peptide is acetylated or free amine; and the peptide is no more than 50 amino acids. As shown herein, such a peptide unexpectedly good nanoparticle stability when complexed with nucleic acid molecules (such as miRNA), which facilitates production, storage, and anti-cancer efficacy of the peptide hydrogel / nanoparticle composite materials described herein.

[0093] The peptide is an amphiphilic cationic peptide that can fold into a [3-hairpin conformation comprising a p-turn, two [3-strands, a hydrophobic face, and a hydrophilic face under appropriate conditions. For example, the peptide folds into an [3-hairpin conformation when dissolved at 2.0% w / v in 50 mM Bis Tris Propane, pH 7.4, 150 mM NaCl, at 25°C, but does not fold into the (3- hairpin conformation when dissolved in water.

[0094] In some aspects, the N-terminus of the first amphiphilic cationic peptide is acetylated. In some aspects, the C-terminus of the first amphiphilic cationic peptide is amidated.

[0095] In several aspects, the first amphiphilic cationic peptide does not contain any negatively charged amino acids, which are believed to disfavor binding to the nucleic acid molecule of the nanoparticle.

[0096] The peptide can be any length appropriate for forming a nanoparticle complex with a nucleic acid molecule. In some examples, the peptide is from about 31 to about 75 residues (e. ., from about 31 to about 50 residues or from about 31 to about 40 residues, “about” refers to plus or minus 2 residues). In some aspects, the peptide can be no more than 50 residues, such as no more than 35 residues or no more than 40 residues. In additional aspects, the peptide can be 31, 35, 40, 45, or 50, residues in length. In some aspects, the peptide can be 31 amino acids in length.

[0097] The peptide can be synthesized using any appropriate technique, such as automated solid phase procedures. The peptide may incorporate one or more modified amino acid residues (e.g., D-amino acids, homologs of naturally occurring amino acids, amino acids with modified side chains, etc.). Exemplary techniques and procedures for peptide syntheses, including solid phase synthesis, are described in Hussein, Skwarczynski, Toth (editors), 2020. Peptide Synthesis: Methods and Protocols, 1sted., Humana. Additional exemplary techniques for peptide synthesis are taught by Bodanszky, M. and Bodanszky, A., The Practice of Peptide Synthesis, Springer Verlag, New York, 1994; and by Jones, J., Amino Acid and Peptide Synthesis, 2nd ed., Oxford University Press, 2002. Peptides of the disclosure can also be readily purchased from commercial suppliers of synthetic peptides once the supplied is provided with the sequence of the peptide.

[0098] Following synthesis, exemplary techniques for peptide purification include reverse phase chromatography, high performance liquid chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, and gel electrophoresis. The actual conditions used to purify a particular peptide, or a modified form thereof, will depend, in part, on synthesis strategy and on factors such as net charge, hydrophobicity, hydrophilicity, and the like.

[0099] C. Nanoparticle-hydrogel composite for nucleic acid delivery

[0100] Provided herein are aspects of a nanoparticle-hydrogel composite material containing a colloidal peptide-nucleic acid molecule nanoparticle dispersed within a peptide hydrogel. The nanoparticle-hydrogel composite displays shear-thin / recovery mechanical properties, which allow the nanoparticle -hydrogel composite and any additional therapeutic dispersed within the hydrogel to be delivered locally to a body cavity, for example, via percutaneous or surgical access by syringe injection, or spray delivery to coat anatomic surfaces. After application, the nanoparticles timerelease from the hydrogel matrix to adjacent tissues and are taken up by cells. Once internalized by cells, the nucleic acid molecule is released from the nanoparticle and (depending on the nucleic acid molecule) may affect cellular function.

[0101] The peptide-nucleic acid molecule nanoparticle comprises a nucleic acid molecule complexed with a first amphiphilic cationic peptide that is not in a [3-hairpin conformation. The peptide hydrogel is formed from a fibrillar network of a second amphiphilic cationic peptides that is in a P-hairpin conformation. The hydrogel can carry other biologic agents (e.g. , encapsulated chemotherapeutics) permitting combinatorial therapies. A discussion of the components of the nanoparticle-hydrogel composite is provided below.

[0102] Nanoparticles and Peptides

[0103] The nanoparticle encapsulated within the peptide hydrogel comprises a nucleic acid molecule complexed with a first amphiphilic cationic peptide that is not in a P-hairpin conformation.

[0104] The first amphiphilic cationic peptide is admixed with the nucleic acid molecule under conditions where the peptide is not in a P-hairpin conformation to form the complex of the nucleic acid molecule and the first amphiphilic cationic peptide (for example, by flash nanocomplexation). The nanoparticle component of the nanoparticle-hydrogel composite can readily be produced, for example, by mixing the first amphiphilic cationic peptide and the nucleic acid molecule in water under conditions sufficient to form the peptide-nucleic acid molecule nanoparticles. Any suitable ratio of the peptide to the nucleic acid molecule can be used that effectively forms the peptide: nucleic acid molecule nanoparticles in aqueous solution. The linear first amphiphilic cationic peptide and the nucleic acid molecule interact via electrostatic interaction between the cationic peptide and anionic nucleic acid to form the nanoparticle.

[0105] Once the nanoparticles are formed, they are mixed with the second amphiphilic cationic P- hairpin peptide under conditions sufficient for the second amphiphilic cationic P-hairpin peptide to fold into a P-hairpin conformation and form a peptide hydrogel that encapsulated the nanoparticles (such as 50 mM Bis Tris Propane, pH 7.4, 150 mM NaCl, at 25°C). Under these conditions and following gelation, the first peptide-nucleic acid molecule complex is a colloidal nanoparticle dispersed within the peptide hydrogel.

[0106] When the nanoparticle is released from the peptide hydrogel and internalized by cells, the first amphiphilic cationic peptide disassociates from the nucleic acid molecule, allowing the nucleic acid molecule to mediate its biological action. It is believed that having the first amphiphilic cationic peptide not folded into a P-hairpin conformation promotes dissociation of the peptide from the nucleic acid molecule after the nanoparticle is internalized by cells.

[0107] The electrostatic charge of the first amphiphilic cationic peptide is equal to or more positive than the electrostatic charge of the second amphiphilic cationic peptide (used to form the peptide hydrogel) at neutral pH. In some aspects, the electrostatic charge of the first amphiphilic cationic peptide is from +7 to +10 (such as +7, +8, +9, or +10) and the electrostatic charge of the second amphiphilic cationic peptide is from +3 to +8 (such as +3, +4, +5, +6, +7, or +8) at neutral pH. In some aspects, the first amphiphilic cationic peptide comprises or consists of an amino acid sequence set forth as:

[0108] KKKKKKKKSGGVKVKVKVKVDPPTKVKVKVKV (PKM1, SEQ ID NO: 1) wherein theDP is a proline that is a D amino acid, the C -terminus of the peptide is amidated or free carboxylic acid, the N-terminus of the peptide is acetylated or free amine; and the peptide is no more than 50 amino acids in length (such as from 25-50 amino acids in length). The peptide folds into an [3-hairpin conformation when dissolved at 2.0% w / v in 50 mM Bis Tris Propane, pH 7.4, 150 mM NaCl, at 25°C, but does not fold into the p-hairpin conformation when dissolved in water.

[0109] In some aspects, the N-terminus of the first amphiphilic cationic peptide is acetylated. In some aspects, the C-terminus of the first amphiphilic cationic peptide is amidated.

[0110] In several aspects, the first amphiphilic cationic peptide does not contain any negatively charged amino acids, which are believed to disfavor binding to the nucleic acid molecule of the nanoparticle.

[0111] Nucleic Acid Molecule Included in the Nanoparticle

[0112] The nucleic acid molecule in the nanoparticle can be any appropriate nucleic acid molecule for delivery to cells in a nanoparticle context. Non-limiting examples include miRNA, plasmid DNA, siRNA, shRNA, long non-coding RNA, mRNA, and dinucleotides.

[0113] In some aspects, the nucleic acid molecule is a dinucleotide, such as 20. The peptide hydrogel of claim 19, wherein the dinucleotide is any one of 2'3'-cGAMP, c-di-GMP, or cAIMP.

[0114] In a non- limiting aspect, the nucleic acid molecule is a plasmid DNA molecule encoding a therapeutic protein (e.g., a protein capable of inducing a desired therapeutic or prophylactic effect when administered to a subject). In some aspects, the therapeutic protein is a vaccine antigen.

[0115] In some aspects the nucleic acid molecule is an antisense nucleic acid molecule, such as a siRNA, shRNA, or antisense miRNA, or a mimic and / or mimetic thereof.

[0116] In a preferred aspect, the nucleic acid molecule is a miRNA, or a or a mimic and / or mimetic thereof. The miRNA or mimic and / or mimetic thereof can be utilized, for example, in methods for treating cancer. miRNAs are small non-coding RNA molecules that regulate gene expression. Mature miRNAs are generally about 17-25 nucleotides in length. miRNAs typically modulate gene expression (e.g., increase or decrease translation) by promoting cleavage of target mRNAs or by blocking translation of the cellular transcript. miRNAs are processed from primary transcripts known as “pri-miRNA” to short stem-loop structures called “precursor (pre)-miRNA.” The pre- miRNA is processed to a miRNA duplex and finally to functional, mature single-stranded miRNA. During processing of the miRNA duplex, one strand (referred to as the “passenger” strand) is degraded, while the other strand (the “guide” strand) is the mature miRNA molecule. Mature miRNA molecules are partially complementary to one or more messenger RNA molecules, and their primary function is to down-regulate gene expression. As disclosed herein, a miRNA nucleic acid includes precursor miRNAs, as well processed or mature miRNA nucleic acids. For example, a miRNA nucleic acid may be a pri-miRNA, a pre-miRNA, a miRNA duplex, or a mature miRNA nucleic acid. miRNA sequences are publicly available. One of ordinary skill in the art can identify miRNA precursors, as well as processed or mature miRNAs, for example, utilizing publicly available databases. For example, miRBase (mirbase.org) includes a searchable database of annotated miRNA sequences. miRNA sequences are also available through other databases known to one of ordinary skill in the art, including the National Center for Biotechnology Information (ncbi.nlm.nih.gov). One of ordinary skill in the art can also identify targets for specific miRNAs utilizing public databases and algorithms, for example at MicroCosm Targets (ebi.ac.uk / enright- srv / microcosm / htdocs / targets / ), TargetScan (targetscan.org), and PicTar (pictar.mdc-berlin.de). Based on miRNA sequences from one organism (such as mouse), one of ordinary skill in the art can utilize the available databases to determine a corresponding miRNA from another organism (such as human).

[0117] In some examples, miRNA functions by activating cleavage or destabilization of a target mRNA or non-coding RNA, which can be detected by RT-PCR, is situ hybridization, FRET, northern blot, or sequencing. It may also function by inhibiting translation of a target mRNA into a protein, which may be detected by Western blot, immune blotting, florescence polarization assay, enzyme activity assay, FRET, immunofluorescence, immunohistochemistry, ELISA, or mass spectrometry. The resulting change in expression of targeted mRNAs or non-coding RNA may result in repression of a number of cancer relevant phenotypes including cell proliferation, resisting cell death, pro-inflammatory processes, increased migration and invasion, angiogenesis, evasion of immune destruction, replicative immortality, decreased genome stability, deregulated cellular energetics, and / or deregulation of epigenetic processes which effect tumor growth and progression.

[0118] In some examples, the nucleic acid molecule of use in the nanoparticle-hydrogel composite and methods disclosed herein include any one of the nucleic acid molecules listed in Table 1. In some examples, the miRNA nucleic acids of use in the nanoparticle-hydrogel composite and methods disclosed herein include the mature miRNAs listed in Table 1. In other examples, the miRNA nucleic acids include those with at least 75% sequence identity to those listed in Table 1 (<?.g., miRNA mimics), as long as such modified miRNAs retain one or more functions of the unmodified miRNA. For example, the nucleic acid molecule (e.g., miRNA) includes or consists of a nucleic acid sequence at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99%, or 100% identical to the nucleic acid sequence of one of the nucleic acid molecules (e.g., miRNAs) listed in Table 1. Additional miRNA nucleic acids of use in the disclosed compositions and methods include a miRNA including guide and / or passenger strands, as long as such modified miRNAs retain one or more functions of the unmodified miRNA. In some examples, the miRNAs with at least 75% sequence identity to those shown in Table 1 , include at least one (such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) non-naturally occurring nucleotide.

[0119] Table 1. Exemplary miRNAs, AntagomiR / miRNA inhibitors, and other nucleotides for use in the disclosed nanoparticle-hydrogel composite.

[0120]

[0121] In some aspects, the nucleic acid molecule included in the nanoparticle -hydrogel composite is selected from any one of the nucleic acid molecule listed in Table 1. In some aspects, the nanoparticle-hydrogel composite comprises a mixture of different nanoparticles comprising two or more (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleic acid molecules selected from the nucleic acid molecules listed in Table 1.

[0122] In some aspects, the nucleic acid molecule included in the nanoparticle-hydrogel composite is a miRNA, or a mimic and / or mimetic thereof, selected from any one of the mature miRNAs listed in Table 1, or a mimic and / or mimetic thereof. In some aspects, the nanoparticle-hydrogel composite comprises a mixture of different nanoparticles comprising two or more (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10) miRNAs, or a mimic and / or mimetic thereof, selected from the mature miRNAs listed in Table 1, or a mimic and / or mimetic thereof.

[0123] In a preferred aspect, the nucleic acid molecule included in the nanoparticle-hydrogel composite is a miRNA selected from any one of hsa-miR-l-5p, hsa-miR-24-1, hsa-miR-26a, hsa- miR-26b, hsa-miR-30b, hsa-miR-130a, hsa-miR-134, hsa-miR-145-5p, hsa-miR-148a, hsa-miR- 149, hsa-miR-192, hsa-miR-194, hsa-miR-206, hsa-miR-215-5p, hsa-miR-342-5p, hsa-miR-370- 5p, has-miR-491-5p, has-miR-497-5p, has-miR-1293, has-miR-5581-5p, hsa-miR-6751-5p, hsa- miR-634, hsa-miR-6842-5p, hsa-miR-6880-5p, hsa-miR-6819-5p, and hsa-miR-4498 , or a mimic and / or mimetic thereof. In some aspects, the nanoparticle-hydrogel composite comprises a mixture of different nanoparticles comprising two or more (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10) miRNAs, or a mimic and / or mimetic thereof, selected from hsa-miR-l-5p, hsa-miR-24-1, hsa-miR-26a, hsa-miR- 26b, hsa-miR-30b, hsa-miR-130a, hsa-miR-134, hsa-miR-145-5p, hsa-miR-148a, hsa-miR-149, hsa-miR-192, hsa-miR-194, hsa-miR-206, hsa-miR-215-5p, hsa-miR-342-5p, hsa-miR-370-5p, has- miR-491-5p, has-miR-497-5p, has-miR-1293, has-miR-5581-5p, hsa-miR-6751-5p, hsa-miR-634, hsa-miR-6842-5p, hsa-miR-6880-5p, hsa-miR-6819-5p, and hsa-miR-4498 , or a mimic and / or mimetic thereof. In a preferred aspect, the nucleic acid molecule included in the nanoparticle -hydrogel composite is a miRNA selected from any one of miR-1, miR-145, miR-206, miR-215-5p, miR-491- 5p, miR-497-5p, miR-1293, miR-634, miR-5581-5p, miR-4498, miR-5681a, miR-3165, miR-342- 5p, and miR-6715b-5p, or a mimic and / or mimetic thereof. In some aspects, the nanoparticlehydrogel composite comprises a mixture of different nanoparticles comprising two or more (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10) miRNAs, or a mimic and / or mimetic thereof, selected from miR-1, miR-145, miR-206, miR-215-5p, miR-491-5p, miR-497-5p, miR-1293, miR-634, miR-5581-5p, miR-4498, miR-5681 a, miR-3165, miR-342-5p, and miR-6715h-5p, or a mimic and / or mimetic thereof.

[0124] In additional examples, the miRNA nucleic acid is longer or shorter than the nucleotide sequence of any one of the miRNA nucleic acids disclosed herein, as long as the miRNA nucleic acid retains a function of the particular miRNA, such as hybridization to a miRNA target sequence or formation of a miRNA duplex. For example, a miRNA nucleic acid can include a few nucleotide deletions or additions at the 5'- or 3 '-end of the nucleotide sequence of a miRNA described herein, such as addition or deletion of 1, 2, 3, 4, or more nucleotides from the 5’- or d’end, or combinations thereof (such as a deletion from one end and an addition to the other end). In particular examples, modified miRNAs described herein include addition of one or more nucleotides at the 3' end, such as addition of one or more nucleotides (for example, 1, 2, 3, or more nucleotides) at the 3' end of a miRNA passenger strand.

[0125] Also provided by the present disclosure are miRNAs that include variations to a disclosed miRNA sequence, as long as such modified miRNAs retain one or more functions of the unmodified miRNA. In some examples, the modifications provide increased stability of a guide strand-passenger strand duplex. In some examples, the modifications include substitutions at one or more nucleotides (such as 1, 2, 3, 4, 5, or more nucleotides) in a miRNA.

[0126] Also provided are miRNA mimetics, such as miRNA nucleic acids that include one or more modified nucleotides or nucleic acid analogs. In some aspects, the isolated miRNA includes at least one nucleobase modification, for example to increase nuclease resistance, enhance half-life and / or improve efficacy. Nucleobase modifications suitable for application to miRNAs are known (see, for example, U.S. Patent Application Publication Nos. 2010 / 0298407; 2007 / 0213292; 2006 / 0287260; 2006 / 0035254; 2006 / 0008822; and 2005 / 0288244).

[0127] In some examples (for example, to increase nuclease resistance and / or binding affinity to a target nucleic acid molecule), a miRNA of the disclosure includes 2’-O-methyl, 2’-fluorine, 2’-O- methoxyethyl, 2’-O-aminopropyl, 2'-amino sugar modifications and / or phosphorothioate linkages. Inclusion of locked nucleic acids (LNA), ethylene nucleic acids (ENA) (<?.g., 2'-4'-ethylene-bridged nucleic acids) and certain nucleobase modifications can also increase binding affinity to the target. The inclusion of pyranose sugars in the oligonucleotide backbone can also decrease endonucleolytic cleavage. Additional modifications include morpholines, peptide nucleic acids (PNA), unlocked nucleic acids (UNA), a-L-LNA, 4'-C-hydroxymethyl-DNA, 2'-N- adamantylmethylcarbonyl-2'-amino-LNA, 2'-N-pyren-l-ylmethyl-2'-amino-LNA, E2'-aminoethyl, 2'-guanidinoethyl, 2'-cyanoethyl, 2'-aminopropyl, oxetane-LNA, 2',4'-carbocyclic-LNA-locked nucleic acid, 2',4'-carbocyclic-ENA-locked nucleic acid, 2'-deoxy-2'-N,4'-C-ethylene-LNA, altritol nucleic acid, hexitol nucleic acid, 2'-aminoethoxymethyl, and 2'-aminopropoxymethyl.

[0128] Additional miRNA mimetics include miRNAs with modified backbones or non-natural intemucleoside linkages. Oligomers having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone are generally referred to in the art as nucleobase oligomers. Nucleobase oligomers that have modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, methyl and other alkyl phosphonates including 3’-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. Various salts, mixed salts and free acid forms are also included. miRNAs having modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.

[0129] In other examples, the modified miRNAs (e.g., miRNA mimetics) include one or more substituted sugar moieties. Such modifications include 2'-O-methyl, 2'-methoxyethoxy, 2'- dimethylaminooxyethoxy, 2'-aminopropoxy, and 2'-fluoro modifications. Modifications may also be made at other positions on an oligonucleotide or other nucleobase oligomer, particularly the 3' position of the sugar on the 3’ terminal nucleotide. Nucleobase oligomers may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. In further examples, a modified miRNA (e.g., a miRNA mimetic) includes a modification at the 5' or 3' end. Such modifications include a primary amino group (for example, with a carbon spacer, such as amino-C3, amino-C6, or amino-C12) at the 5' end of the miRNA. Additional end modifications include UNAs, methylphosphonate, phosphithorate, an inverted base, or an N- methyl-G cap.

[0130] In other aspects, the miRNA includes two or more modifications, such as two or more modifications selected from a base substitution, a modification at an internucleoside linkage, a modified sugar, or a modification at the 5' and / or 3' end. For duplex miRNA molecules, the modification(s) may be present on the guide strand, the passenger strand, or both.

[0131] In some examples, the modified e.g., mimic or mimetic) miRNA nucleic acids disclosed herein include a 5' end amino modification, such as a 5'-amino C6 modification (such as a 5'-amino C6 modified passenger strand). In other examples, the modified e.g., mimic or mimetic) miRNA nucleic acid includes one or more nucleotides (such as 1, 2, 3, 4, 5, 6, 7, 8, or more nucleotides) with a 2' modification (such as 2'-0-Me). The 2' modified nucleotides may be internal to the miRNA (none of the modifications are on the 5' or 3' end nucleotide) or may include the 5' and / or 3' end nucleotides. In some examples, a miRNA guide strand includes one or more nucleotides (such as 3-10, 4-9, or 5-8 nucleotides) having a 2' modification. In specific examples, a guide strand includes 2' modifications on one or more internal nucleotides, and in some examples, not on a 5' or 3' end nucleotide. In other examples, a miRNA passenger stand includes one or more nucleotides (such as 3-10, 4-8, or 5-7 nucleotides) having a 2' modification. In specific examples, a passenger strand includes 2' modifications on a 5' or 3' end nucleotide, but may also include 2' modification of one or more internal nucleotides.

[0132] In some aspects, the disclosed miRNA nucleic acids or modified (e.g., mimetic or mimic) miRNA nucleic acids are associated with a detectable label. In some examples, the miRNA nucleic acid is conjugated to a fluorescent label (such as fluorescein isothiocyanate, coumarin, Cy3, Cy5, Cy7, or Alexa Fluor® dyes), a hapten (such as digoxigenin or Myc), or a radioactive label. In other aspects, the miRNA nucleic acid is associated with a peptide or protein (for example, to facilitate targeted delivery), such as tat, MACV GP1, folate receptor, penetratin, mesothelin, or epidermal growth factor receptor. One of skill in the art can select additional detectable labels or peptides depending on the particular circumstances.

[0133] In some aspects, the nucleic acid molecule included in the nanoparticle is a plasmid DNA molecule encoding one or more of the disclosed miRNA nucleic acids or a mimic or mimetic thereof. The miRNA nucleic acid or a mimic or mimetic thereof encoded by plasmid DNA molecule can be operably linked to any suitable promoter for expression. Suitable promoters for expressing RNA from a plasmid include, for example, the U6 or Hl RNA pol III promoter sequences, a cytomegalovirus promoter, an SV40 promoter or metallothionein promoter. Selection of other suitable promoters is within the skill in the art. The recombinant plasmids can also comprise inducible or regulatable promoters for expression of the miRNA gene products. In one non-limiting aspect, the miRNA nucleic acid is expressed as an RNA precursor molecule from a plasmid, and the precursor molecule is processed into a functional or mature miRNA within the target cell.

[0134] Peptide Hydrogel

[0135] The peptide hydrogel component of the nanoparticle-hydrogel composite material is formed from a fibrillar network of the second amphiphilic cationic peptide that is in a P-hairpin conformation.

[0136] The P-strand regions of the hairpin contain alternating sequences of hydrophobic (e.g., valine) and hydrophilic (charged) residues e.g., lysine) such that in the folded state, one face e.g., the valine-rich face) of the peptide is hydrophobic and the opposing face (e.g., the lysine rich face) is lined with positively charged side chains and is hydrophilic. This amphiphilic arrangement facilitates inter-molecular peptide interactions, and the fibril arrangement necessary for hydrogel formation.

[0137] Self-assembly is facilitated facially by hydrophobic association of the hydrophobic faces of folded hairpins and laterally via H-bond formation and hydrophobic van der Waals contacts between neighboring hairpins. Detailed knowledge of these parameters allows control the selfassembly process and thus the ultimate hydrogel material properties. For example, under folding conditions peptides may adopt a desired secondary structure (e.g. , may adopt an amphiphilic - hairpin structure where one face of each P-strand in the hairpin is lined with hydrophobic residues and the other face is lined with hydrophilic residues). For example, intramolecular folding is dictated by the alleviation of charge density on the hydrophilic face upon folding, the formation of intramolecular hydrophobic van der Waals interactions, the formation of intramolecular hydrogen bonds between -strands within the hairpin, and the turn propensity of the P-turn sequence included in the peptide.

[0138] Thus, peptides for use in the hydrogel component of the nanoparticle-hydrogel composite can be constructed to have desired characteristics by varying one or more of at least the following parameters: 1) electrostatics, for example, by varying the charge within the peptide intramolecular folding and self-assembly rates can be varied; 2) van der Waals interactions, for example, constructing peptides having varying a) lateral and facial intermolecular hydrophobic interactions and / or b) intramolecular hydrophobic interactions, allows varying the folding and self-assembly of the peptides as well as the material properties of the hydrogel; 3) hydrogen bonding, for example peptides may be constructed with varying a) intramolecular and / or b) intermolecular hydrogen bond formation to vary the folding, self-assembly and final material properties; and 4) turn sequence, for example, the turn region of peptides of the invention may be designed to control folding and thus trigger self-assembly.

[0139] The peptide includes high P-sheet propensity residues flanking an intermittent four residue turn sequence. Polar and apolar residues may be arranged sequentially in the strand regions to afford amphiphilic surfaces when the peptide is folded in a P-hairpin conformation. In the disclosed PKM 1 peptide, these four residues are VDPPT, and the type II’ P-turn is defined by the dihedral angles (Phi and Psi) adopted by theDPP portion of the turn sequence, where ‘D’ denotes D- stereochemistry of the first proline residue. The preferred Phi and Psi dihedral angles (degrees) that define a type II’ turn are: residue i+1 (60,-120); residue i+2 (-80,0). However, these values can vary by + / - 20 degrees and the peptide can still form the appropriate P-turn structure.

[0140] Similarly, HLT2, a 20-residue peptide is composed of high P-sheet propensity valine, glutamate, and serine residues flanking an intermittent tetrapeptide -VDPPT- designed to adopt type-II' -tum structure.

[0141] In addition to incorporating local design elements to stabilize hairpin structure, nonlocal effects were also considered by arranging the polar and apolar residues flanking the P-turn in an alternating fashion to favor P-hairpin formation in the self-assembled state. In addition, a P~ branched residue was placed at the i-position of the Lum to enforce a trans prolyl amide bond geometry at the i+1 position. This design element ensures that under folding conditions, intramolecular folding of monomeric hairpins is favored prior to self-assembly. A cis prolyl bond, which is designed against, could result in the presentation of individual P-strands within each monomer in an extended conformation. Peptides capable of adopting both cis and trans conformers could undergo indiscriminant self-association of extended and correctly folded monomers and may be actively designed against.

[0142] In some aspects, the second amphiphilic cationic peptide comprises or consists of an amino acid sequence set forth as: VLTKVKTKVDPPTKVEVKVLV (HLT2, SEQ ID NO: 2), wherein theDP is a proline that is a D amino acid, the C-terminus of the second peptide is amidated or free carboxylic acid, the N-terminus of the second peptide is acetylated or free amine, and the second peptide is no more than 50 amino acids in length. The peptide folds into an P-hairpin conformation when dissolved at 2.0% w / v in 50 mM Bis Tris Propane, pH 7.4, 150 mM NaCl, at 25°C. In some aspects, the N-terminus of the second amphiphilic cationic peptide is acetylated and the C-terminus of the second amphiphilic cationic peptide is amidated.

[0143] The second amphiphilic cationic peptide can fold into a P-hairpin conformation comprising a p-turn. two P-strands, a hydrophobic face, and a hydrophilic face under appropriate conditions (e.g., 2.0% w / v peptide in 50 mM Bis Tris Propane, pH 7.4, 150 mM NaCl, at 25°C). Under the appropriate conditions, the second amphiphilic cationic peptide self-assembles into a fibrillar network wherein the peptide is folded in an P-hairpin conformation in the fibrillar state.

[0144] The nanoparticle-hydrogel composite including a peptide hydrogel based on the second cationic amphiphilic peptide in a P-hairpin confirmation can readily be made by preparing an aqueous solution comprising one or more of the second cationic amphiphilic peptides (such as HLT2) and nanoparticle (such as a PKMl:miRNA particle) as disclosed herein and altering one or more characteristics of the solution, wherein a hydrogel is formed. The characteristic altered may be any characteristic that results in formation of a hydrogel upon its alteration. Suitable examples include, but are not limited to, ionic strength, temperature, concentration of a specific ion, and pH. In particular aspects, the character altered may be the pH of the solution. The second cationic amphiphilic peptide forms a hydrogel at a pH of about 7 or higher. Increasing pH and increasing ionic strength both encourage hydrogel formation, and the two effects are roughly additive. Thus, the lower the pH, the higher the salt concentration necessary for hydrogel formation. In some aspects, the hydrogel can be formed in a container (such as a syringe), for example a closed container.

[0145] In some aspects, altering one or more characteristic of the solution results in a salt concentration of from about 10 mM to about 400 mM, such as about 50 to about 300 mM, about 100 to about 200 mM, or about 150 mM. Any salt may be used, for example, KC1, NaCl, MgCh, KF, MgSCU. etc. In one aspect, the salt may be NaCl. In some aspects, the solution may have a desired pH, for example, a pH of from about 7 to about 9, a pH of from about 7.5 to about 8.5, a pH of from about 7.0 to about 8.0, or a pH of about 7.4, which may stay the same or be changed upon formation of the hydrogel.

[0146] In one non- limiting example, the hydrogel is formed in 50 mM Bis Tris Propane (BTP), 150 mM NaCl, pH 7.4. Any buffer system can be used except phosphate based buffer systems, as phosphate buffers are known to precipitate P-hairpin peptides. Accordingly, peptide hydrogels including second cationic amphiphilic peptide can simply be formed by, for example, adding buffer of appropriate ionic strength to an aqueous solution of unfolded peptide; drawing the resulting solution into a syringe; and allowing it to gel at 25 °C directly in the syringe. The nanoparticle-hydrogel composite is a well hydrated solid material having a stiffness greater than 40 Pascal (Pa), as measured by the storage modulus G' at a strain of 0.2%. Above approximately 40 Pa the material is a self-supporting solid gel material. The stiffness can reach greater than 10,000 Pa at higher peptide concentration. The nanoparticle-hydrogel composite typically contains at least 0.25 wt % of the second amphiphilic cationic peptide in an aqueous medium. For example, the nanoparticle-hydrogel composite may be formed comprising a percent by weight of second amphiphilic cationic peptide of from about 0.25% w / v to about 4.0% w / v, from about 0.25% w / v to about 3.0% w / v, from about 0.25% w / v to about 2.0% w / v, from about 0.25% w / v to about 1.0% w / v, from about 0.5% w / v to about 4.0% w / v, from about 0.5% w / v to about 3.0% w / v, from about 0.5% w / v to about 2.0% w / v, from about 0.5% w / v to about 1.0% w / v, from about 1.0% w / v to about 4.0% w / v, from about 1.0% w / v to about 3.0% w / v, from about 1.0% w / v to about 2.0% w / v, from about 2.0% w / v to about 4.0% w / v, or from about 2.0% w / v to about 3.0% w / v.

[0147] In one aspect, the amount by weight of the second amphiphilic cationic peptide and the kinetics of gelation may be varied to produce a nanoparticle-hydrogel composite having a desired modulus (stiffness). Hydrogels of the invention may have a modulus from about 40 Pascal (Pa) to about 50,000 Pa, from about 40 Pa to about 25,000 Pa, from about 40 Pa to about 10,000 Pa, from about 40 Pa to about 5,000 Pa, from about 40 Pa to about 1,000 Pa, from about 40 Pa to about 500 Pa, from about 40 Pa to about 100 Pa, from about 100 Pa to about 50,000 Pa, from about 100 Pa to about 25,000 Pa, from about 100 Pa to about 10,000 Pa, from about 100 Pa to about 5,000 Pa, from about 100 Pa to about 2,000 Pa, from about 100 Pa to about 1,000 Pa, from about 100 Pa to about 500 Pa, or from about 100 Pa to about 250 Pa.

[0148] The resultant nanoparticle-hydrogel composite is mechanically rigid and displays shear- thinning / recovery behavior. This characteristic provides a free flowing suspension during the application of shear and complete reformation of the gel network (self-healing) after cessation of the shear. This combination of shear thinning and self-healing allows material formation in a spatially resolved manner. For example, in some aspects, a pre-formed nanoparticle-hydrogel composite can be injected or sprayed (shear thin) into a target location in a subject where it self heals and reforms the nanoparticle-hydrogel composite. The shear stress converts the gel to a lower viscosity, flowable fluid. The shear stress is relieved when the fluid exits the syringe or spray nozzle and the gel quickly self-heals, recovering its original mechanical rigidity. This shear- thinning / recovery mechanism allows the nanoparticle-hydrogel composite to be easily delivered by syringe or spray to the target location in the subject. The first and second amphiphilic cationic peptides for use as disclosed herein can be any length appropriate for forming a nanoparticle complex with a nucleic acid molecule and for forming a peptide hydrogel. In some examples, the first and second peptides are from about 20 to about 75 residues {e.g., from about 20 to about 50 residues, from about 20 to about 40 residues, from about 20 to about 30 residues, from about 20 to about 25 residues, from about 20 to about 50 residues, from about 20 to about 40 residues, from about 20 to about 30 residues, or from about 20 to about 25 residues (“about” refers to plus or minus 2 residues). In some aspects, the peptides for use in the disclosed aspects can be from 20 to 75 residues (e.g., from 20 to 50 residues, from 20 to 40 residues, from 20 to 30 residues, from 20 to 25 residues, from 20 to 50 residues, from 20 to 40 residues, from 20 to 30 residues, or from 20 to 25 residues). In some aspects, the peptide can be no more than 50 residues, such as no more than 30 residues or no more than 20 residues. In additional aspects, the peptide can be 20, 25, 30, 35, 40, 45, or 50, residues in length. In some aspects, the peptide can be 20 amino acids in length.

[0149] The first and second amphiphilic cationic peptides for use in the disclosed aspects can be synthesized using any appropriate technique, such as automated solid phase procedures. The first and second amphiphilic cationic peptides may incorporate one or more modified amino acid residues (e.g., D-amino acids, homologs of naturally occurring amino acids, amino acids with modified side chains, etc.). Exemplary techniques and procedures for peptide syntheses, including solid phase synthesis, are described in Hussein, Skwarczynski, Toth (editors), 2020. Peptide Synthesis: Methods and Protocols, 1s‘ ed., Humana. Additional exemplary techniques for peptide synthesis are taught by Bodanszky, M. and Bodanszky, A., The Practice of Peptide Synthesis, Springer Verlag, New York, 1994; and by Jones, J., Amino Acid and Peptide Synthesis, 2nd ed., Oxford University Press, 2002. Peptides of the disclosure can also be readily purchased from commercial suppliers of synthetic peptides once the supplied is provided with the sequence of the peptide.

[0150] Following synthesis, exemplary techniques for peptide purification include reverse phase chromatography, high performance liquid chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, and gel electrophoresis. The actual conditions used to purify a particular peptide, or a modified form thereof, will depend, in part, on synthesis strategy and on factors such as net charge, hydrophobicity, hydrophilicity, and the like.

[0151] Additional Agents Encapsulated within the Nanoparticle-Hydrogel Composite

[0152] In some aspects, the disclosed nanoparticle-hydrogel composite includes one or more heterologous agents dispersed within the hydrogel. For example, in some aspects, the nanoparticle-hydrogel composite includes one or more heterologous anti-cancer agents dispersed within the hydrogel. Non-limiting examples of anticancer agents include a cytokine, a chemokine, an antibody, or a chemotherapeutic agent. The cytokine can be, for example, interleukin-2 (IL-2), granulocyte macrophage colony stimulating factor (GM-CSF), or interferon, such as interferon (IFN) p. In some aspects, the antibody is a PD-1 antagonist, such as antibody that specifically binds PD-1 or PD-L1, such as MPDL3280A.

[0153] Non- limiting examples of chemotherapeutic agents that can be included in the nanoparticlehydrogel composite include, but are not limited to alkylating agents, such as nitrogen mustards (for example, chlorambucil, chlormethine, cyclophosphamide, ifosfamide, and melphalan), nitrosoureas (for example, carmustine, fotemustine, lomustine, and streptozocin), platinum compounds (for example, carboplatin, cisplatin, oxaliplatin, and BBR3464), busulfan, dacarbazine, mechlorethamine, procarbazine, temozolomide, thiotepa, and uramustine; antimetabolites, such as folic acid (for example, methotrexate, pemetrexed, and raltitrexed), purine (for example, cladribine, clofarabine, fludarabine, mercaptopurine, and thioguanine), pyrimidine (for example, capecitabine), cytarabine, fluorouracil, and gemcitabine; plant alkaloids, such as podophyllum (for example, etoposide, and teniposide), taxane (for example, docetaxel and paclitaxel), vinca (for example, vinblastine, vincristine, vindesine, and vinorelbine); cytotoxic / antitumor antibiotics, such as anthracycline family members (for example, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin), bleomycin, hydroxyurea, and mitomycin; topoisomerase inhibitors, such as topotecan and irinotecan; monoclonal antibodies, such as alemtuzumab, bevacizumab, cetuximab, gemtuzumab, rituximab, panitumumab, and trastuzumab; photosensitizers, such as aminolevulinic acid, methyl aminolevulinate, porfimer sodium, and verteporfin; and other agents , such as alitretinoin, altretamine, amsacrine, anagrelide, arsenic trioxide, asparaginase, bexarotene, bortezomib, celecoxib, denileukin diftitox, erlotinib, estramustine, gefitinib, hydroxycarbamide, imatinib, pentostatin, masoprocol, mitotane, pegaspargase, and tretinoin.

[0154] In a particular example, for a nanoparticle-hydrogel composite for use in treating mesothelioma, the heterologous anti-cancer agent included in the nanoparticle-hydrogel composite comprises pemetrexed, cisplatin, bevacizumab, carboplatin, gemcitabine, vinorelbine, or a combination of two or more thereof.

[0155] In a particular example, for a nanoparticle-hydrogel composite for use in treating cervical cancer, the heterologous anti-cancer agent included in the nanoparticle-hydrogel composite comprises cisplatin, fluorouracil, paclitaxel, bevacizumab, tototecan, carboplatin, gemcitabine, or a combination of two or more thereof. In a particular example, for a nanoparticle-hydrogel composite for use in treating a thymoma or thymic carcinoma, the heterologous anti-cancer agent included in the nanoparticle- hydrogel composite comprises cisplatin, doxorubicin, cyclophosphamide, prednisone, vincristine, etoposide, ifosfamide, paclitaxel, or a combination of two or more thereof.

[0156] In a particular example, for a nanoparticle-hydrogel composite for use in treating ovarian cancer, the heterologous anti-cancer agent included in the nanoparticle-hydrogel composite comprises paclitaxel, cisplatin, carboplatin, docetaxel, bevacizumab, or a combination of two or more thereof.

[0157] In a particular example, for a nanoparticle-hydrogel composite for use in treating non-small cell lung cancer, the heterologous anti-cancer agent included in the nanoparticle-hydrogel composite comprises paclitaxel, cisplatin, carboplatin, docetaxel, bevacizumab, pemetrexed, etoposide, gemcitabine, vinorelbine, or a combination of two or more thereof.

[0158] In a particular example, for a nanoparticle-hydrogel composite for use in treating non-small cell lung cancer, the heterologous anti-cancer agent included in the nanoparticle-hydrogel composite comprises paclitaxel, cisplatin, carboplatin, docetaxel, bevacizumab, pemetrexed, etoposide, gemcitabine, vinorelbine, or a combination of two or more thereof.

[0159] In a particular example, for a nanoparticle-hydrogel composite for use in treating esophageal cancer, the heterologous anti-cancer agent included in the nanoparticle-hydrogel composite comprises paclitaxel, carboplatin, oxaliplatin, fluorouracil, or a combination of two or more thereof.

[0160] The amphiphilic peptides disclosed herein are cationic. Accordingly, in typical aspects involving a heterologous agent encapsulated within the peptide hydrogel, the heterologous agent has a neutral or net positive charge to prevent binding of the agent to the hydrogel matrix. Depending on the agent, the neutral or net positive charge may lead to varying retention time in the peptide hydrogel.

[0161] Peptide hydrogels including a heterologous agent can be readily produced by preparing an aqueous solution comprising the heterologous agent (such as a chemotherapeutic agent) and the second amphiphilic P-hairpin peptide (such as HLT2) and nanoparticle (such as a MAX 1 :miRNA particle) as disclosed herein and altering one or more characteristics of the solution, wherein a hydrogel is formed. As discussed above, the characteristic altered may be any characteristic that results in formation of a hydrogel upon its alteration, such as ionic strength, temperature, concentration of a specific ion, and pH. In some aspects, the hydrogel including the heterologous agent can be formed in a container (such as a syringe), for example a closed container. The peptide hydrogel including an encapsulated heterologous agent can be used for any suitable purpose. For example, peptide hydrogels with an encapsulated heterologous agent can be administered to a subject in need thereof (for example, by injection to a target location in the subject).

[0162] D. Treatment and Prevention of Cancer

[0163] It is disclosed herein that administration of the disclosed nanoparticle-hydrogel composite (for example, including HLT2-hased hydrogel and PKM 1 :hsa-miR-215 nanoparticles) inhibits the growth and metastasis of tumors in vivo. This observation supports the use of the disclosed nanoparticle-hydrogel composites as therapeutics for the treatment an inhibition of cancer.

[0164] Accordingly, methods are disclosed herein for treating or inhibiting cancer in a subject by administrating an effective amount of disclosed nanoparticle-hydrogel composite to the subject. The nanoparticles in the composite comprise a nucleic acid molecule that has an anti-cancer effect when introduced into cells. For example, the nucleic acid molecule can be a miRNA, or a mimic and / or mimetic thereof, with anti-cancer activity, or a plasmid DNA vector encoding a miRNA, or a mimic and / or mimetic thereof, with anti-cancer activity. In particular examples, the methods include administering to a subject with cancer an effective of a nanoparticle-hydrogel composite comprising nanoparticles containing one or more miRNAs that are down-regulated in the cancer, in order to treat or inhibit the cancer in the subject. In another example, the methods include administering to a subject with cancer an effective of a nanoparticle-hydrogel composite comprising nanoparticles containing one or more anti-miRs (miRNA Inhibitors) that specifically bind to miRNAs that are up-regulated in the cancer, in order to treat or inhibit the cancer in the subject.

[0165] In some aspects, the methods include treating an existing cancer in a subject. In additional aspects, methods are disclosed herein are used for preventing metastasis of a cancer in a subject.

[0166] Subjects that can benefit from the disclosed methods include humans and veterinary subjects. A suitable administration format may be determined by a medical practitioner for each subject individually.

[0167] Subjects can be screened prior to initiating the disclosed therapies, for example to determine whether the subject has cancer, such as a serosal neoplasm (e.g., mesothelioma). The presence of the cancer in the subject indicates that the cancer can be treated using the methods provided herein. The presence of a cancer in a subject can be determined by methods known in the art, and typically include cytologic, morphologic, or molecular-based evaluation. The cancer can be one with an established tumor. The cells of the cancer that are screened can be in vivo or ex vivo, including cells obtained from a biopsy. In some aspects, a subject can be selected for treatment that has, is suspected of having, or is at risk of developing, cancer, a serosal neoplasm (e.g., mesothelioma).

[0168] The cancer treated by the methods disclosed herein can be any cancer of interest, including, but not limited to, a serosal neoplasm (e.g., mesothelioma). Non-limiting examples of cancers that can be treated using the disclosed methods include skin cancers, breast cancers, brain cancers, cervical carcinomas, testicular carcinomas, head and neck cancers, gastrointestinal tract cancers, genitourinary system cancers, gynecological system cancers, endocrine system cancers, a sarcoma of the soft tissue and bone, a mesothelioma, a melanoma, and a neoplasm of the central nervous system. In some aspects, the cancer is a head and neck cancer, such as cancers of the nasal cavity, paranasal sinuses, nasopharynx, oral cavity, oropharynx, larynx, hypopharynx, salivary glands, and paragangliomas. In other aspects, the cancer is a lung cancer, such as a non-small cell lung cancer or a small cell lung cancer. In further aspects, the cancer can be a cancer of the gastrointestinal tract, such as cancer of the esophagus, stomach, pancreas, liver, biliary tree, small intestine, colon, rectum and anal region. In yet other aspects, the cancer can be a cancer of the genitourinary system, such as cancer of the kidney, urethra, bladder, prostate, urethra, penis and testis. In some aspects, the cancer is a gynecologic cancer, such as cancer of the cervix, vagina, vulva, uterine body, gestational trophoblastic diseases, ovarian, fallopian tube, peritoneal, or breast. In other aspects, the cancer is an endocrine system cancer, such as a thyroid cancer, parathyroid cancer, adrenal cortex cancer, pancreatic endocrine cancer, carcinoid cancer and carcinoid syndrome. The cancer can be a sarcoma of the soft tissue and bone, a mesothelioma, a cancer of the skin, a melanoma, comprising cutaneous melanomas and intraocular melanomas, a neoplasm of the central nervous system, a cancer of the childhood, comprising retinoblastoma, Wilm's tumor, neurofibromatoses, neuroblastoma, Ewing's sarcoma family of cancers, rhabdomyosarcoma. The cancer can be a lymphoma, comprising non-Hodgkin's lymphomas, cutaneous T-cell lymphomas, primary central nervous system lymphoma, and Hodgkin's disease. The cancer can be plasma cell neoplasms, a cancer of unknown primary site, a peritoneal carcinomastosis, a Kaposi's sarcoma, AIDS-associated lymphomas, AIDS -associated primary central nervous system lymphoma, AIDS- associated Hodgkin’s disease and AIDS-associated anogenital cancers, a metastatic cancer to the liver, metastatic cancer to the bone, malignant pleural and pericardial effusions and malignant ascites. In specific non-limiting examples, the cancer is a serosal neoplasm (e.g. , mesothelioma).

[0169] In some non-limiting aspects, the method includes treating or inhibiting a cancer present or at risk of being present at a serosal surface (e.g., part of a serosal body cavity) lined by mesothelial cells in the subject. In some non-limiting aspects, the method include treating or inhibiting a cancer present or at risk of being present in a serosal body cavity in the subject, such as a pleural cavity, a pericardial cavity, an anterior mediastinal cavity, a posterior mediastinal cavity, a peritoneal cavity, or a tunica vaginalis testis cavity. In some aspects, the cancer is a serosal neoplasm, such as a pleural mesothelioma, a peritoneal mesothelioma, a thymic epithelial cancer (i.e. a thymoma, a thymic carcinoma), an ovarian carcinoma, a cervical cancer, a small-cell lung carcinoma, a nonsmall-cell lung carcinoma, an ovarian carcinoma, or an appendiceal cancer.

[0170] In a preferred aspect, the disclosed methods are used to treat mesothelioma in a subject. Treatment of the mesothelioma can reduce a symptom of the mesothelioma in the subject. Symptoms include respiratory symptoms, such as coughing, coughing up blood, wheezing and / or shortness of breath, systemic symptoms such as weight loss, fever, or fatigue, or symptoms due to local compression, such as chest pain, bone pain, or difficulty swallowing. Generally, the methods include selecting a subject having a mesothelioma, and administering to the subject a therapeutically effective amount of a disclosed nanoparticle-hydrogel composite. Treatment of the mesothelioma is generally initiated after the diagnosis of the cancer. A subject with any stage of mesothelioma can be treated using the method disclosed herein. The presence of mesothelioma can be determined by methods known in the art, such as a chest x-ray, CT scan, a PET scan, MRI and / or endobronchial ultrasound. Pulmonary function tests can also be used. The mesothelioma can also be diagnosed by obtaining one or more biopsies and evaluating the cells in the biopsy.

[0171] Treatment of the cancer is generally initiated after the diagnosis of the cancer, or after the initiation of a precursor condition (such as metaplasia or dysplasia). Treatment can be initiated at the early stages of cancer, for instance, can be initiated before a subject manifests symptoms of a condition, such as during a stage I diagnosis or at the time dysplasia is diagnosed. However, treatment can be initiated during any stage of the disease, such as but not limited to stage I, stage II, stage III and stage IV cancers. In some examples, treatment is administered to a subject with a pre- cancerous tumor that can convert into a malignant or even metastatic tumor.

[0172] Treatment initiated after the development of a condition, such as malignant cancer, may result in decreasing the severity of the symptoms of one of the conditions, or completely removing the symptoms, or reducing metastasis, tumor volume or number of tumors. In some example, the cancer becomes undetectable following treatment.

[0173] In one aspect of the disclosure, the formation of tumors in the treated subject, such as metastasis, is delayed, prevented or decreased. In another aspect, the size of the primary tumor in the treated subject is decreased. In a further aspect, a symptom of the tumor is decreased. In yet another aspect, tumor volume is decreased.

[0174] Treatment prior to the development of the condition, such as treatment upon detecting dysplasia or an early (benign) precursor condition, is referred to herein as treatment of a subject that is “at risk” of developing the condition. Administration to a target location in the subject where the cancer is “at risk of being present” refers to administration to a target location in the subject where a cancer has not formally been detected, but where there is a possibility of current cancer, or a possibility of developing a current cancer within a suitable time frame (such as within one year). This includes a site of dysplasia or an early (benign) precursor condition, and / or a site of pre-operative, intra-operative, or post-operative surgical treatment (such as tumor resection) of a cancer, for example, where there is concern that the cancer may not have been fully removed by the surgical procedure.

[0175] In some examples, treatment using the methods disclosed herein prolongs the time of survival of the subject (e.g., increases survival time by at least 6 months, at least 9 months, at least 12 months, at least 2 years, at least 3 years, or even at least 5 years relative to the absence of the therapy).

[0176] One skilled in the art can readily determine an effective amount of a disclosed nanoparticlehydrogel composite to be administered to a subject, for example, taking into account factors such as the type of tumor being treated, the extent of disease progression, the age, health and sex of the subject, the size (e.g. , weight and / or height) of the subject, and the route of administration. For example, the effective amount can be based on the approximate body weight of a subject to be treated. Such effective amounts can be administered by any suitable route. In some aspects, the effective amount of the nanoparticle-hydrogel composite can be based on the amount / concentration of the nanoparticles dispersed within the nanoparticle-hydrogel composite containing therapeutic nucleic acid molecules.

[0177] An effective amount of the nanoparticle-hydrogel composite is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer. In one aspect, an effective amount of the nanoparticle- hydrogel composite is the amount necessary to inhibit tumor growth (such as growth of a lung tumor), metastasis of the tumor, or the amount that is effective at reducing a sign or a symptom of the tumor. The effective amount of the agents administered can vary depending upon the desired effects and the subject to be treated. In some examples, an effective amount is an amount that eliminates or reduces the patient's tumor burden, or that prevents or reduces the growth of metastatic cells.

[0178] In some examples, an effective amount of a disclosed nanoparticle-hydrogel composite comprises miRNA nucleic acid (or combination of miRNA nucleic acids) of from about 5 pg / kg to about 100 mg / kg of body weight, such as about 100 pg / kg to about 10 mg / kg, about 1 mg / kg to about 20 mg / kg, about 1 mg / kg to about 25 mg / kg, about 20 mg / kg to about 40 mg / kg, about 30 mg / kg to about 50 mg / kg, or about 40 mg / kg to about 100 mg / kg. In one non-limiting example, the amount administered is about 5 mg / kg of a miRNA nucleic acid (or a combination of miRNA nucleic acids). In some examples, an effective amount of a disclosed nanoparticle-hydrogel composite comprises miRNA nucleic acid (or combination of miRNA nucleic acids) of from about 1 mg / kg to about 20 mg / kg.

[0179] The particular mode of administration and the dosage regimen will be selected by the attending clinician, taking into account the particulars of the case (e.g. the subject, the disease, the disease state involved, and whether the treatment is prophylactic). The nanoparticle-hydrogel composite can be formulated in unit dosage form, suitable for individual administration of precise dosages. The amount of active compound(s) administered will be dependent on the subject being treated, the severity of the affliction, and the manner of administration, and is best left to the judgment of the prescribing clinician. Within these bounds, the nanoparticle-hydrogel composite to be administered will contain a quantity of the active component(s) in amounts effective to achieve the desired effect in the subject being treated. Multiple treatments are envisioned, such as over defined intervals of time, such as daily, bi-weekly, weekly, bi-monthly or monthly, such that chronic administration is achieved. Administration may begin whenever appropriate as determined by the treating physician.

[0180] The disclosed nanoparticle-hydrogel composite can be administered to a subject in need of treatment using any suitable means. Methods of administration include, but are not limited to, intraductal, intradermal, intramuscular, intraperitoneal, intrapleural, subcutaneous, vaginal, rectal, intranasal, inhalation. One of skill in the art can select an appropriate route of administration, depending on the therapeutic agent(s), the condition being treated, the health and treatment history of the subject, and other relevant clinical factors.

[0181] The nanoparticle-hydrogel composite displays shear-thin / recovery mechanical properties, which allow the nanoparticle-hydrogel composite and any additional therapeutic dispersed within the hydrogel to be delivered locally to a body cavity, for example, via percutaneous or surgical access by syringe injection, or spray delivery to coat anatomic surfaces. After application, the nanoparticles time-release from the hydrogel matrix to adjacent tissues and are taken up by cells. Once internalized by cells, the nucleic acid molecule is released from the nanoparticle and (depending on the nucleic acid molecule) may affect cellular function.

[0182] In the case of mesothelioma, the nanoparticle hydrogel composite could be sprayed via a hand-held device or through the tip of a minimally invasive surgical instrument during the conduct of surgical resection or diagnosis of this disease. Surgical approaches include open thoracotomy, VATS (video-assisted thoracoscopic surgery), and / or robotic-assisted thoracoscopic surgery. Administration onto the parietal and visceral body surfaces of the composite material could occur before planned surgical resection of tumor or after surgical resection. Depending on the clinical scenario, the nanoparticle hydrogel composite could be applied in this manner repeatedly.

[0183] In some aspects, nanoparticle -hydrogel composite is delivered to a serosal surface (e.g., part of a serosal body cavity) in the subject where the cancer is present or is at risk of being present by direct syringe injection or spray delivery. For example, the serosal surface can be part of a pleural cavity, a pericardial cavity, an anterior mediastinal cavity, a posterior mediastinal cavity, a peritoneal cavity, or a tunica vaginalis testis cavity in the subject. Administration of the nanoparticle-hydrogel composite in this way coats all or a portion of the serosal surface in the subject where the cancer is present or is at risk of being present.

[0184] The present disclosure also includes methods of treating a subject with combinations of the nanoparticle -hydrogel composite (containing a therapeutic nucleic acid molecule) with one or more other agents useful in the treatment of a cancer. For example, the nanoparticle-hydrogel composite can be administered in combination with effective doses of one or more heterologous anti-cancer agents (such as a chemotherapeutic agent or anti-cancer protein) dispersed within the hydrogel. A skilled clinician can select an appropriate combination of therapies based on the type of tumor being treated, the subject’s clinical history, overall condition, and other factors.

[0185] The following examples are provided to illustrate particular features of certain aspects of the disclosure, but the scope of the claims should not be limited to those features exemplified.

[0186] EXAMPLES

[0187] Nanoparticles are an effective delivery strategy for therapeutic nucleic acids, especially for RNAs, as evidenced by the recent success of lipid nanoparticles (NPs) as delivery vehicles of messenger RNA (mRNA) for COVID- 19 vaccination. While many different nanomaterials can package RNA into non-viral vectors by various mechanisms, polyelectrolyte complexation is a highly facile means to form RNA-loaded NPs with high translational potential. Here, supercharged cationic species are introduced to negatively charged RNA, where the interacting molecules complex and condense into NPs after charge neutralization; however, NPs formulated this way face the same challenges as other materials for clinical translation, such as reproducible size and uniformity and aggregation potential. The following examples describe investigation of an engineered microRNA-based polyelectrolyte complex (PEC) NPs for applications in cancer treatment, such as mesothelioma cancer treatment. The miRNA-peptide PEC NPs were formulated to load into a self-assembling peptide-based surface-fill hydrogel (SFH), which can be subsequently sprayed to tissue and locally release the therapeutic NPs (FIG. 1A). These NPs are separately prepared before incorporation into the gel network. As shown herein, many tested peptides resulted in metastable NPs, where their size distribution begins to broaden over time if not quickly encapsulated into the gel phase, thereby complicating transition to large-scale, clinical settings. However, the newly designed PKM1 peptide is shown to form highly stable nanoparticles with miRNA, which facilitates production, storage, and anti-cancer efficacy of the peptide hydrogel / nanoparticle composite materials described herein.

[0188] EXAMPLE 1: MATERIALS AND METHODS Peptide Synthesis and Purification

[0189] Following standard Fmoc-based solid phase synthesis techniques, all peptides were synthesized using a CEM Liberty Blue™ microwave-assisted peptide synthesizer (CEM) and ProTide Rink amide resin (0.58 mmol g1loading) at a 0.25 mmol (431 mg) scale. All amino acids, resin, and oxyma were purchased from CEM Corporation. Solvents and other coupling reagents (DMF, DCM, DIC, HCTU, DIPEA, NMP, piperidine, TFA, and TIS) were purchased from Sigma and Fischer Scientific. All Fmoc deprotection cycles were conducted with 20% piperidine in DMF (4 mL) using a standard microwave deprotection method (15 s at 75 °C) followed by 50 s at 90°C. After deprotection, the resin was washed 4 times with DMF for 5 min per wash. All coupling reactions for amino acids (unless stated otherwise) were performed with Fmoc-protected amino acids (5 eq, 0.2 M in DMF), oxyma (5 eq, 1 M in DMF), and DIC (5 eq, 0.45 M in DMF). The coupling reactions were heated at 90°C for 4 min. For all peptides following coupling of their respective D-proline residue, the subsequent valine residue was triple-coupled at room temperature with HCTU (5 eq, 0.45 M in DMF) and DIPEA (10 eq, 2 M in NMP) for 30 min each. The next two residues (lysine and valine) were double coupled (30 min each) using the same HCTU and DIPEA conditions. The following residues were coupled following the DIC and oxyma conditions. Cleavage of peptides from dried resin was achieved by addition of a cleavage cocktail containing 95% v / v trifluoroacetic acid (TFA), 2.5% v / v triisopropylsilane (TIS), and 2.5% v / v water and stirring for 3 h (10 mL). After 3 h, the cleavage solution was collected and excess TFA was removed by flushing with argon gas at room temperature. The crude peptide was precipitated with cold diethyl ether, dried under vacuum, dissolved in water, frozen, and then lyophilized before purification.

[0190] All peptides were purified by semi-preparative reverse-phase high performance liquid chromatography (RP-HPLC) using a Waters 600 system equipped with a Waters 2489 UV detector and a Vydac 218TP Cl 8 column (250 x 22 mm, 10 pm) at a flow rate of 8.0 mL min1and a column temperature of 40°C with monitoring at 220 nm for peptide absorbance. Mobile phases for RP-HPLC consisted of the solvent system of standard A (STD A, 0.1% v / v TFA in water) and standard B (STD B, 0.1% v / v TFA in 90% v / v acetonitrile / 10% v / v water). Crude peptides were dissolved in STD A at ~4 mg mL1and injected into the Waters 600 system at 5 mL per run. Gradients and elution profiles for each specific peptide are described as follows: for MAXI, SSP1, and 0KM1, isocratic gradient from 0 to 5 min at 5% STD B, a linear gradient from 5% to 13% STD B for 2 min, then a linear gradient from 13% to 100% STD B over an additional 174 min (peptides eluting at -28% STD B); for TSS1, isocratic gradient from 0 to 5 min at 5% STD B, a linear gradient from 5% to 17% STD B for 2 min, then a linear gradient from 17% to 100% STD B over an additional 166 min (peptide eluting at -32% STD B); for PKM1, isocratic gradient from 0 to 5 min at 5% STD B, a linear gradient from 5% to 12% STD B for 2 min, then a linear gradient from 12% to 100% STD B over an additional 176 min (peptide eluting at -27% STD B); for HLT2, isocratic gradient from 0 to 5 min at 5% STD B, a linear gradient from 5% to 20% STD B for 2 min, then a linear gradient from 12% to 100% STD B over an additional 160 min (peptide eluting at -36% STD B). The collected product-containing fractions were combined and lyophilized using a FreeZone -80°C 4.5 Plus freeze dryer (Labconco Corp.). Further characterization of purified peptides was conducted by analytical RP-HPLC and liquid chromatography-mass spectroscopy (LC-MS) to assess purity and molecular weight. Analytical RP-HPLC was performed on an Agilent 1200 series HPLC with a Vydac 218TP C18 column (250 x 4.6 mm, 5 pm) running a linear gradient from 0 to 100% STD B at a rate of 1% STD B per min (flow rate of 1 mL min1) and column temperature of 40°C. Mass spectra were obtained on a Shimadzu LCMS 2020 system using a Luna C18 column 100 A (150 x 3 mm, 5 pm) and solvents consisting of 0.1% v / v formic acid in water (STD A) and 0.1% v / v formic acid in acetonitrile (STD B). Peptides were run on the column under a linear gradient from 0% to 90% STD B at a rate of 2% STD B per min and a column temperature of 40°C. Analytical RP-HPLC chromatograms and ESI (+) mass spectra of pure peptides are provided in Figures S1-S6.

[0191] Preparation of miRNA-Peptide Polyelectrolyte Complex Nanoparticles (PEC NPs)

[0192] All NPs were formulated with chemically modified double- stranded scramble miRNA mimics (mirVana™ miRNA mimic Negative Control #1, catalog # 4464061, Thermo Fisher) or fluorescently-labeled scramble miRNA mimics (both FAM- or Cy3-conjugated depending on the experiment; FAM™-labeled Pre-miR™ Negative Control #1, catalog # AM17121, Thermo Fisher; Cy3™ Dye-labeled Pre-miR Negative Control #1, catalog # AM17120, Thermo Fisher). For miRNA-215-5p (miR-215)-based NPs, miR-215 was obtained from Integrated DNA Technologies (IDT). All NPs were formulated with different concentrations of miRNA and charge ratios depending on the experiment. The specific methodology for NP preparation is detailed below for the two techniques used in this study:

[0193] By bulk mixing: An equal volume of peptide (at 2x final concentration, typically 1 mL) in RNase-free water is pipetted into a solution of miRNA in RNase-free water (at 2x final concentration, typically 1 mL) into RNase-free microcentrifuge tubes (2 mL, Eppendorf). The mixture was then immediately vortexed for 30 s; after which, a 5 min stabilization is applied. The resulting NP suspension is then ready for further use and analysis.

[0194] By flash nanocomplexation (FNC): The FNC preparation of miRNA-peptide PEC NPs was facilitated by a confined impinging jet (CIJ) mixer device (Holland Applied Technologies, catalog # P0288404), which has chamber dimensions that are the same as those previously reported by Prud’homme and Macosko (Johnson and Prud’homme, Physical Review Letters 2003, 91, 118302; Markwaiter et al., JoVE 2019, e58757; J. Han et al., Journal of Pharmaceutical Sciences 2012, 101, 4018). The inlet ports of the CIJ mixer device were fitted with Leur-Lock adapters (IDEX part # P- 604). To allow for attachment of Leur-Lock syringes, the mixer inlet ports were connected to silicone tubing (Bio-Rad catalog # 7318211, 1.6 mm inner diameter, 0.8 mm wall thickness) outfitted with Luer-Lock adapters on either end (IDEX part # P-857 and P-850 on each side). The side ports of the device were plugged with screwable flat-bottom plugs (IDEX part # P-309), which were kept in during CIJ mixer operation. The outlet of the mixing chamber was connected by screw and ferrule (IDEX part # P-205 and P-200X, respectively) to a short (~3 cm in length) EFTE tubing (IDEX part # 1517, 1 mm inner diameter, 1 / 16 in outer diameter) to aid collection of NPs as they exit the mixer. Respective solutions of miRNA or peptide were loaded into sterile, air-tight syringes (Norm-JECT® Leur-Lock syringes (typically 2 mL of each solution per syringe), at sizes ranging from 5 mL to 50 mL (depending on flow rate requirements of the syringe pump) and attached to the inlet tubing of the CIJ mixer. The syringes were then loaded onto a programmable high-pressure dual-syringe pump (NE-4000 SyringeTWO™, New Era Pump Systems). The two solutions were then impinged into the mixing chamber at a fixed flow rate set on the syringe pump (ranging from 0.1 to 50 mL / min). Formulated NPs were collected from the outlet stream into RNase-free microcentrifuge tubes (2 mL, Eppendorf), where the first 0.5 mL was discarded to account for ramp-up time in reaching the internal mixing condition and steady-state. A 5 min stabilization was applied before further analysis or use of the NP suspensions.

[0195] The dynamics of the inlet fluids and their mixing efficiency is characterized by the Reynold’s number Re) to reflect laminar or turbulent flow in the CIJ mixer, which is a dimensionless number representing the ratio of inertial to viscous forces for a given system of fluid(s). For the CIJ mixer, the overall Re number is calculated by accumulating the contribution of multiple streams (Johnson and Prud'homme, AIChE Journal 2003, 49, 22641; Santos et al., Small, 2016, 12, 6214):

[0196] Here, p\ is the density (kg m’3) of the fluid in the zth inlet stream (assumed to be that of water for all inlet streams), Qi is the flow rate of the zth inlet stream (m3s’1), pi is the dynamic viscosity (Pa s) of the fluid of the zth inlet stream (assumed to be that of water for all inlet streams), d is the diameter of the inlet nozzle (m), and n is the number of streams. The device contains two inlet streams to the internal mixing chamber.

[0197] For the characterization of the characteristic mixing time (w) in the CIJ device at different flow rates, we used scaling model for TM that is equivalent to diffusion across the Kolmogorov microscale, as derived and defined by Prud’homme and coworkers (and describe din the examples below). In our calculations, we are assuming that the presence both miRNA and peptide do not influence the density and kinematic viscosity of the inlet streams (which is valid given their low volume fraction). We use the following equation to calculate the characteristic mixing time in the CIJ device:

[0198] Here, KCIJ is a scaling factor based on mixer geometry (-171 for our geometry, empirically-derived and determined), v is the kinematic viscosity of the outlet stream (m2s’1), is the dimensionless intemozzle separation of the mixing chamber (= 5 for our mixer geometry), d is the inlet jet stream diameter (m), u is the inlet jet stream velocity (m s’1), and D is mixing chamber diameter (m). The mixing time was calculated for a series of flow rates used to impinge NP starting materials into the CIJ device.

[0199] Physical Characterization of PEC NPs

[0200] Dynamic Light Scattering: Particle size measurements were conducted by DLS using a Malvern Zetasizer Nano-ZS ZEN3600 (Malvern Panalytical) with light scattering measured with a backscatter angle of 173° and automatic measurement duration settings. All particle suspensions were analyzed in water (water selected as dispersant for measurement) at their formulation concentration with no further dilution and measurements were conducted in disposable ZEN0040 cuvette (UV-transparent cuvette micro, catalog # CEL1062, Scientific Laboratory Supplies Ltd.) at T = 25 °C. Three measurements were made per sample and averaged to determine the Z- average hydrodynamic diameter (Z>z, from cumulants fit analysis) of the system. Particle size distributions were assessed from the intensity-based size distributions. Size standard deviation, as defined in DLS ISO 22412, was used to evaluated particle uniformity, relating the polydispersity index (PDI) to Dz with the equation below:

[0201] Zeta Potential Measurements'. Particle zeta potential was measured using a Malvern Zetasizer Nano-ZS ZEN3600 (Malvern Panalytical) by phase analysis light scattering. Electrophoretic mobility was related to zeta potential under the Smoluchowski approximation. All particle solutions were analyzed in water at their formulation concentration with no further dilution, where 800 pL of sample was loaded into a disposable folded capillary cell (DTS1070 cuvette, Malvern). Samples were run at T = 25 °C with automatic measurement duration, and three measurements were made per sample and averaged. miRNA Encapsulation Efficiency: To determine the total miRNA encapsulated within a given NP formulation, a Quant-iT™ RiboGreen® RNA Reagent Kit (R11490, Invitrogen™, Thermo Fisher Scientific) was used following the manufacturer’s protocol. Samples were transferred to black Nunc™ 96- well polystyrene plates (Thermo Scientific™) and RiboGreen® reagent (100 pL) then added, mixed, and allowed to incubate for 5 min at room temperature. The plates were then analyzed in a BioTek Synergy Neo2 multi-mode microplate reader (Gen5 v3.08 data analysis software) to measure fluorescence emission intensity (Xex= 485 nm,em= 530 nm; gain = 90). Fluorescence intensity was normalized to free scrambled miRNA (at the concentration for the given NP formulation) in RNase-free water and background corrected.

[0202] Circular Dichroism Spectroscopy

[0203] All CD spectroscopy experiments were conducted with a Jasco J- 1500 Circular Dichroism Spectrophotometer with Spectra Manager software (version 2.14.06). All miRNA-peptide nanoparticles were prepared as described above in RNase-free water, maintaining a constant peptide concentration of 35 pM across all tested designs (with each respective charge ratio). Each free peptide or miRNA-peptide solution (200 pL) was transferred to a 1 mm path length quartz cuvette and run. Continuous wavelength scans were carried out between 260 and 190 nm at a 1 nm step size (D.I.T. of 2 s, bandwidth of 1 nm, scan rate of 50 nm min1) and T = 25°C. Raw sample spectra were corrected for miRNA and background contribution and then converted to mean residue ellipticity, [0] (deg cm2dmol '), using the following equation:

[0204] Where $obs is the measured ellipticity (mdeg), I is the path length of the cell (0.1 cm), c is the molar concentration of the peptide (M), and r is the number of amino acid residues of the peptide. NP Binding Isotherms & Competitive Decomplexation Assay

[0205] For the fluorescence binding isotherms conducted for each peptide design and scramble miRNA (Figure 3B), equal volume solutions of peptide and scramble miRNA (50 pL) in RNase- free water were mixed by bulk mixing (as described above) at molar ratios ranging from 0.1 to 500 for peptide:miRNA (at a fixed concentration of miRNA, 0.5 pg ml / 1). The amount of miRNA encapsulated by each peptide at each concentration was determined using a Quant-iT™ RiboGreen® RNA Reagent Kit (R1 1490, Tnvitrogen™, Thermo Fisher Scientific) following the manufacturer’s protocol. Samples were transferred to black Nunc™ 96- well polystyrene plates (Thermo Scientific™) and RiboGreen® reagent (100 pL) then added, mixed, and allowed to incubate for 5 min at room temperature. The plates were then analyzed in a BioTek Synergy Neo2 multi-mode microplate reader (Gen5 v3.08 data analysis software) to measure fluorescence emission intensity (kcx= 485 nm, ZLm= 530 nm; gain = 90). Fluorescence intensity was normalized to free scrambled miRNA (0.5 pg ml1) in RNase-free water and background corrected.

[0206] For competition-induced decomplexation assessment of miRNA-peptide nanoparticles (Figure 1 J , S12. S15), NPs were formed at a charge ratio (N / P) of 10 for all peptides with 10 pg mL1scrambled miRNA by flash nanocomplex ation (unless noted as bulk mixing otherwise) in RNase-free water. To each sample of NPs (100 pL), heparin sulfate (10 pL, Heparin sodium salt from porcine intestinal mucosa, Grade I-A, Sigma- Aldrich) in RNase-free water was added at increasing concentrations from 0.1 to 10000 pg mL1(at lOx concentration to account for dilution factor). The NP and heparin mixtures were then incubated at 37°C and agitated for 1 h at 60 rpm. The amount of miRNA decomplexed from the peptide for each sample was determined by a Quant- iT™ RiboGreen® RNA Reagent Kit (R11490, Invitrogen™, Thermo Fisher Scientific) following the manufacturer’s protocol. Samples were transferred to black Nunc™ 96-well polystyrene plates (Thermo Scientific™) and RiboGreen® reagent (100 pL) then added, mixed, and allowed to incubate for 5 min at room temperature. The plates were then analyzed in a BioTek Synergy Neo2 multi-mode microplate reader (Gen5 v3.08 data analysis software) to measure fluorescence emission intensity (kex= 485 nm, Xem= 530 nm; gain = 90). Fluorescence intensity was normalized to samples of free scrambled miRNA with heparin sulfate in RNase-free water and background corrected.

[0207] Transmission Electron Microscopy

[0208] Carbon-coated copper grids (400-square mesh, CF400-CU; Electron Microscopy Science, Inc.) were treated with plasma air for 30 s using a PELCO easiGlow™ glow discharge cleaning system (Ted Pella, Inc.) to render the carbon film more hydrophilic. A 10 pL droplet of NPs was then deposited on top of a plasma-treated grid and allowed to sit for 1 min. Then, the excess solution was wicked away with filter paper, leaving a thin film of the sample atop the grid. A 7 pL droplet of uranyl acetate (2 wt% in MilliQ water) was then added on top of the grid for negative staining and blotted away after 30 s. Grids were allowed to dry for at least 3 h before imaging on an FEI Tecnai 12 Twin transmission electron microscope operating at 80 kV acceleration voltage. All images were recorded using an AMT XR16 charge-coupled device (CCD) bottom- mount camera (Advanced Microscopy Techniques, Corp.). NP diameters were measured using ImageJ software.

[0209] Cell Culture

[0210] Human mesothelioma cell line H2052 was obtained from ATCC. Low passage, patient- derived mesothelioma cell line #52 (MB52) was obtained from Mesobank UK, an international bioresource (Chernova et al., Cell Death Differ. 2016, 23, 1152; ALTaei et al., Lung Cancer 2012, 77, 312). Mesothelioma cell lines were cultured as monolayers at 37 °C and 5% CO2 in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin solution.

[0211] Cell Viability

[0212] Mesothelioma cells were seeded into 96- well plates at a density of 3.0 x 103cells / well 24 hours before treatment of nanoparticles. After 96 hours of treatment, cell viability was measured using the CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega). Studies were performed in three independent cell preparations.

[0213] In Vitro Transfection Efficiency

[0214] Mesothelioma cells were seeded at 2 x 10scells / well into 6-well plates 24 hours before nanoparticle treatment. The FNC-formulated FAM-labeled scrambled miRNA-peptide nanoparticles (N / P = 3 for PKM1 -based nanoparticles, N / P = 10 for other peptides) were diluted with optiMEM media (Gibco, Thermo Fisher Scientific) to achieve a final miRNA concentration of 40 nM and then incubated with cells for 2 hours. After incubation, cells were washed with PBS, trypsinized (5 min) and collected by centrifugation. Cell pellets were washed and resuspended in 500 pL of PBS for flow cytometry analysis. Data were acquired on FACSCalibur™ flow cytometer (BD LSRII; data acquisition, FACSDiva v.8.0.1) and analyzed using FlowJo vl0.8 (BD Biosciences). Complexes of commercially available transfection agent Lipofectamine RNAiMAX (Thermo Fisher Scientific), and equivalent amount of FAM-labeled scrambled miRNA were prepared according to the manufacturers’ instructions and analyzed in the same conditions as nanoparticles.

[0215] Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

[0216] Total RNA was isolated from cells using TRIzol™. Quantification of RNA was done using a NanoDrop 2000 spectrophotometer. For mRNA analysis, total RNA was reverse-transcribed by using the High-Capacity cDNA Reverse Transcription Kit. For miRNA analysis, reverse transcription was performed using the TaqMan advanced miRNA cDNA synthesis kit. Expression of mRNA or miRNA was determined by TaqMan analysis on a QuantStudio 6 Flex PCR system. The expression of gene and miRNA was normalized to beta-actin and U6 (RNU6B) small- nucleolar RNA, respectively. qRT-PCR primers used for the analysis of genes and miRNA expression were available from Applied Biosystems (see the following table). All qRT-PCR reactions were performed independently in triplicate. All reagents and equipment were obtained from Thermo Fisher Scientific.

[0217] Preparation of Surface-Fill Hydrogel

[0218] Solutions of miRNA-peptide nanoparticles were prepared as described above in RNase-free water. For a 200 p L formulation of the SFH, the suspension of NPs (50 pL, 4x concentration) are added to a solution of ice-chilled hydrogelating peptide HLT2 (50 pL, 4x concentration). To this mixture, ice-chilled 2xHBS buffer (100 pL; 50 mM HEPES, 300 mM NaCl, pH 7.4) is added and gently mixed. This final mixture is incubated at T = 37°C overnight, affording 200 pL of SFH (final concentrations of lx miRNA and lx HLT2) in IxHBS buffer (25 mM HEPES, 150 mM NaCl, pH 7.4) loaded with miRNA-peptide NPs. Depending on the experiment, these volumes and concentrations are adjusted to formulate the SFH.

[0219] In Vitro Release of NPs from SFH

[0220] SFH samples (100 pL final volume) were prepared as described above into cylindrical glass vials (12 x 35 mm, half dram, Fisher Scientific) for a total of 1 pg of FAM-labeled scrambled miRNA per gel (10 pg mL1, formulated into nanoparticles with MAXI (N / P = 10), TSS1 (N / P = 10), or PKM1 (N / P = 3) peptides) with 0.5 wt% (or 1 wt%) HLT2 gel in IxHBS buffer (25 mM HEPES, 150 mM NaCl, pH 7.4). After overnight curing at T = 37°C, 1 mL of IxHBS buffer was added to the top of the gels, and the vials were incubated at T = 37°C under constant agitation (60 rpm). At the scheduled time points, the entire supernatant was removed from the top of the gel and replaced with 1 mL of fresh IxHBS buffer. To ensure complete miRNA dissociation from released nanoparticles, 100 pL of heparin sulfate (Heparin sodium salt from porcine intestinal mucosa, Grade LA, Sigma- Aldrich) in RNase-free water (1 mg mL1) was added to the removed supernatants at each time point. The supernatant and heparin mixtures were then incubated at T = 37°C and agitated at 60 rpm for 1 h before further analysis. Afterward, the concentration of released miRNA in the supernatant was determined by measuring the fluorescence signal of FAM- miRNA and calculating the % released from a standard curve. For the FAM-miRNA, samples were excited at 490 nm with emission intensity monitored from 400 to 600 nm (1 nm step size, 0.5 s integration, 5 nm slit widths, Xcm,max = 520 nm) on a Horiba PTIQuantaMaster-400 fluorimeter (FelixGX 4.2.2. software).

[0221] Oscillatory Shear Rheology

[0222] Rheological characterization of 1 wt% SFH (300 pL) containing miRNA-peptide nanoparticles (10 pg mL'1scrambled miRNA formulated with MAXI (N / P = 10), TSS1 (N / P = 10), or PKM1 (N / P = 3)) was conducted on a TA Instruments HR20 rheometer (Trios v5.7.0.526 software) using a 25 mm stainless steel parallel plate geometry (gap height = 0.5 mm). SFH samples were prepared in 5 mL NORM-JECT® Luer-Lock syringes on top of the syringe plunger (with Luer-Lock portion removed by razor blade) and cured overnight at 37 °C wrapped with parafilm. At time of analysis, SFH samples were transferred from the syringe to the center of the rheometer plate, which was pre-equilibrated at 37 °C, and the geometry was then lowered to the gap height (0.5 mm). A dynamic time-sweep experiment was then performed, monitoring storage and loss moduli for 60 min at a constant angular frequency (6 Rad s’1) and fixed 0.2% strain at 37°C, after which, the samples were subjected to 1000% strain for 30 s (at 6 Rad s’1frequency, 37°C) with followed by recovery monitored for 60 min (6 Rad s’1, 0.2% strain, 37°C). After the dynamic time-sweep, dynamic frequency sweeps (increasing frequency from 0.1 to 100 Rad s’1at constant 0.2% strain) and amplitude sweeps (increasing strain from 0.1 to 1000% at constant 6 Rad s’1frequency) were conducted to ensure time-sweep experiments were conducted in the linear viscoelastic regime and to determine flow strain of the gels, respectively. For SFH formulations containing lyophilized nanoparticles, the final cryoprotectant content in the gel is 0.625% w / v D- mannitol. In Vivo Mesothelioma Xenograft Model

[0223] All animal experiments were approved by our Animal Care and Use Committee in accordance with NIH Guidelines. NOD.Cg-PrWcsc,d7Z2rzm7W77 / SzJ (NSG) mice (6-8 weeks old) were used to generate human subcutaneous mesothelioma xenograft (Shultz et al., J Immunology 2005, 174, 6477).

[0224] Biodistribution: Briefly, 3xlO6MB52 cells stably expressing the luciferase reporter vector pGL4.51 [Iuc2 / CMV / Neo] (Promega) were subcutaneously injected into the right flank of NSG male mice. Xenograft growth was continuously monitored over 4 weeks by measuring the tumor volume as well as tumor luminescence using IVIS imaging system (PerkinElmer Inc., Waltham, MA). To determine the tumor uptake and tissue distribution of miRNA particles released from SFH, 200 pL of SFH (1 wt% HLT2, IxHBS buffer, pH 7.4) loaded with cyanine 3(Cy3)-labeled scrambled miRNA-MAXl or -PKM1 nanoparticles, and empty SFH control (100 pg / mL Cy3-miR concentration in gel, n = 3 mice / group) was injected peritumorally when tumors reached an average volume of -100 mm34 weeks post-injection of cells. Treatment group of mice injected with empty SFH was considered as a as control to correct tissue autofluorescence. Whole-body images of mice were acquired at day 0 and 4 post-SFH administration to monitor Cy3 fluorescence and tumor luminescence under IVIS imaging system. On day 4, mice were sacrificed; tumors and vital organs were collected and imaged. Living Image software (version 4.3.1) was used for image acquisition and analysis. A total of 200 pL (15 mg / mL) of D-luciferin potassium salt solution (Regis Technologies) was administered into the intraperitoneal cavity of mice immediately before imaging at the indicated time points.

[0225] Histofluorescence: To evaluate the intratumoral localization of released Cy3-miRNA nanoparticles from implanted SFH, frozen tumor tissues were used to generate 5-pm-thick sections that were fixed with 4% formaldehyde in PBS. Tissue sections were mounted with a medium containing DAPI (Vector Laboratories) to stain the nuclei and imaged using KEYENCE fluorescence microscope (BZ-X800E, KEYENCE, USA).

[0226] Tumor Growth Inhibition: For the anti-tumor efficacy tumor efficacy of SFH loaded with miRNA-peptide PEC nanoparticles, MB52 cells (3.0xl06cells / mouse) stably expressing luciferase were injected subcutaneously into the right flank of NSG. After 4 weeks, when xenografts establishment was confirmed by measuring tumor luminescence and volume (reached an average volume of -120 mm3), mice were randomized and divided into four treatment groups (n=5 for each group). Mice were peritumorally administered with 200 pL of each SFH formulation (scr-miR- MAX1, miR-215-MAXl, scr-miR-PKM 1 , miR-215-PKMl nanoparticles) with nanoparticles containing 20 pg miRNA (100 pg / mL concentration in gel). Tumor growth was monitored over a period of 4 weeks at the indicated time point by measuring luminescence using IVIS imaging system. Tumor progression was also monitored by measuring the tumor volume at indicated time points.

[0227] In Vivo Gene Silencing-. NSG mice bearing subcutaneous MB52 tumor xenografts with an average volume of -100 mm3were peritumorally injected with 200 pL of each SFH formulation (scr-miR-MAXl, miR-215-MAXl, scr-miR-PKMl, miR-215-PKMl nanoparticles, n=4 mice for each group). One-week post-SFH administration, mice were sacrificed, and tumors collected. Total RNA was isolated from resected tumors using a standard protocol. Both miRNA-215 levels and gene expression were analyzed using quantitative PCR as described before in qRT-PCR section.

[0228] Freeze-Drying, Cryopreservation, and Reconstitution of NPs

[0229] The freeze-drying of NP formulations (prepared as described above with different peptides and miRNA concentrations, depending on the experiment) was conducted on a FreeZone -80°C 4.5 Plus freeze dryer (Labconco Corp.) with vacuum levels operating at 0.013 to 0.021 mbar (T = - 80°C). Prior to putting samples on the lyophilizer, samples were either frozen in a -80°C freezer or flash-frozen by submerging samples in liquid nitrogen until completely frozen. Samples were allowed to dry on the freeze dryer for 12 h. Following drying, lyophilized powders of NPs were then stored at specified storage condition (4°C or -80°C) until specified time points, where particles were reconstituted using RNase-free water and subsequently analyzed or used (depending on the experiment).

[0230] For samples that were cryoprotected, sterile, molecular biology grade sucrose (Thermo Scientific), trehalose (AMSBIO), and D-mannitol (Sigma-Aldrich) 2x stock solutions were prepared in RNase-free water (to account for dilution to lx after adding to NPs). Equal volume of cryoprotectant stock (typically 1 mL) was added to a given NP suspension (typically 1 mL) and gently mixed to achieve a 1 : 1 dilution (yielding final cryoprotectant concentrations ranging from 2.5 to 10% w / v, depending on sample and experiment). Samples were then immediately frozen at - 80°C or with liquid nitrogen and lyophilized following the procedure stated above. Reconstitution was conducted with RNase-free water at room temperature with an equal volume to the initial volume before drying (for 1 mL NPs with 1 mL cryoprotectant, 2 mL water is used for reconstitution) and gentle mixing. EXAMPLE 2: miRNA-PEPTIDE PEC ASSEMBLY

[0231] Turbulent Micromixing to Control miRNA-Peptide PEC Assembly

[0232] The preparation of uniform, stable nanoparticles is dependent on the diffusion rates of starting materials that define their mixing. Diffusion rates dictate local and transient concentration profiles during mixing and assembly, which defines how the complexation reaction initiates, propagates, and terminates (Hu el al., ACS Nano 2019, 13, 10161 ; Storkle el al., Macromolecules 2007, 40, 7998). Large polyelectrolyte macromolecules typically exhibit slower diffusion rates compared to their electrostatic complexation rates. Further, the diffusion rates can be vastly different between polyelectrolyte pairs of the formulation. For example, given the size difference between miRNA-215-5p and the peptides studied herein (~14 kDa vs ~2 kDa), their diffusion rates are likely to be quite different. Achieving kinetic control over mixing affords control of molecular diffusion and thus the mechanism by which particles are formed. miRNA-Peptide Polyelectrolyte Complex Nanoparticles miRNA-peptide particle formation likely occurs in two steps (FIG. 1C). First, positively charged peptide diffuses into the vicinity of miRNA and binds, neutralizing charge and forming a PEC chain. This step is defined by the time regime called the characteristic mixing time (TM). Second, individual PEC chains condense and associate with neighboring chains to form the nanoparticle. This second step is often rate limiting and defined as the characteristic assembly time (T ). When miRNA-peptide NPs are formed by conventional bulk mixing, the events in these two time regimes overlap, resulting in heterogenous particle formation. Kinetic control over these time regimes is only possible when homogenous mixing of starting materials is faster than their assembly into NPs. Homogenous mixing occurs when the characteristic mixing time (TM) is significantly shorter than the characteristic assembly time (TA) (Johnson et al., AIChE Journal 2003, 49, 2264; Johnson et al., Australian Journal of Chemistry 2003, 56, 1021 ; Johnson et al., Physical Review Letters 2003, 91, 118302; York et al., Advanced Materials 2012, 24, 733). Since TA is largely dependent on the physical and chemical properties intrinsic to the polyelectrolytes involved, for example, nucleic acid type and length, peptide size and structure, and the stoichiometry of miRNA-peptide binding, this parameter is predetermined and thus difficult to change (Hu et al., ACS Nano 2019, 13, 10161). On the other hand, TM can be precisely varied using turbulent micromixing within a CIJ mixer, where shortened diffusion paths of the components achieves values of TM sufficiently shorter than TA (Johnson et al., AIChE Journal 2003, 49, 2264). Thus, by employing fast CIJ mixer inlet jet velocities, these otherwise overlapping time regimes can become distinct. This results in nearly instantaneous homogenous mixing of all miRNA and peptide molecules, resulting in the formation of PEC chains before any subsequent chain condensation begins.

[0233] CIJ mixer performance was first characterized using a scaling model for TM that is equivalent to diffusion across a relevant length scale characteristic of the mixing energy input into the system, as derived and defined by Prud’homme and coworkers (Johnson et al., AIChE Journal 2003, 49, 2264; Johnson et al., Australian Journal of Chemistry 2003, 56, 1021 ; Johnson et al., Physical Review Letters 2003, 91, 118302). With this model, the single length scale that adequately characterizes the fluid striation distribution formed within the mixer is the Kolmogorov microscale, which refers to the smallest eddy dimension that can form turbulence prior to viscous effects dominating and yielding a laminar flow microstructure (Johnson et al., AIChE Journal 2003, 49, 2264). The Kolmogorov microscale is obtained faster relative to molecular diffusion with momentum diffusion serving as the active mixing mechanism, not molecular diffusivity (Johnson et al., AIChE Journal 2003, 49, 2264). With the assumption that both inlet streams and the outlet stream from the CIJ mixer have equivalent densities and based on the mixer geometry used in this study, a proportionality relationship for TM is given by Equation (1) below:

[0234] Here, Kcu is a scaling factor based on mixer geometry, v is the kinematic viscosity of the outlet stream, A is the ratio of the mixing chamber diameter to the inlet jet stream diameter, d is the inlet jet stream diameter, and u is the inlet jet stream velocity. By this relationship, enhanced mixing (shorter TM) can be achieved with greater turbulence generated (i.e., reducing the Kolmogorov length scale) by impinging at higher flow rates into the CIJ mixer chamber. First, the TM necessary to achieve kinetic control of the assembly of model scrambled miRNA was determined with the cationic peptide MAXI (FIGs. 9A-9B). MAXI is a 20 residue amphiphile containing 8 lysine residues and a free terminal amine, resulting in a charge of +9 at neutral solution pH. All the peptides used in this study were synthesized by standard Fmoc-based solid phase peptide synthesis, purified by reverse-phase high-performance liquid chromatography, and characterized by liquid chromatography mass spectroscopy (FIGs. 9A-9B, 10A-10B, 11A-11B, 12A-12B, 13A-13B, 14A- 14B). The scrambled miRNA is a chemically modified double-stranded miRNA mimic that bears a -45 charge at neutral solution pH. The inlet jet flow rates carrying these components into the CIJ mixer were varied (at a fixed charge ratio of N / P = 10), and the relationship above was used to calculate (see methods in Example 1) (Johnson et al., Physical Review Letters 2003, 91, 118302). The effect of inlet jet flow rate, Q, was assessed on both particle size and uniformity as measured by dynamic light scattering (DLS) where uniformity is reflected in size standard deviation and correlated to its TM (FIGS. 2A-2B). Two distinct regions are observed where particle size and uniformity transition from being ^-independent to <9-dependent. In the first region at short TM (high Q), all formulated particles measure the same size with a similar degree of uniformity, and these values are constant regardless of Q. In this region, the mixing is extremely efficient and not diffusion- limited, resulting in TM < TA- Here, PEC chain formation occurs before chain condensation and association, which yields consistently sized and highly uniform NPs. In the second region at long M (low Q), NPs are larger and less uniform, and these factors are dependent on and inversely proportional with Q. This region likely represents scenarios where TM > TA and mixing is diffusionlimited. As such, PEC chain formation, condensation, and association events occur roughly together to produce a heterogeneous population of particles. The effects of mixing on NP uniformity is further illustrated by observed particle size distributions, where mixing at high Q (short TM) yields narrow distributions (FIG. 2C). Moreover, the transition between these two regions coincides with a transition from laminar (low Q) to turbulent (high Q) flow, and thus a transition from macromixing (like that of conventional bulk mixing) to micromixing (FIG. 15). In selecting the optimal Q for production, it is important to consider that by increasing the concentration of miRNA, the TA for the system decreases, as observed by the transition point between regions occurring at comparatively shorter values of TM (FIGS. 2A-2B). Given this consideration, kinetic control was achieved at < 55 ms, and therefore 20 mL min1was selected as an optimal flow rate for FNC production and subsequent studies.

[0235] Flash Nanocomplexation Outperforms Bulk Mixing

[0236] With the optimal FNC processing parameters determined, the NP characteristics of miRNA- MAX1 NPs formulated by FNC to was compared to the bulk mixing methodology (Majumder et al., Nature Nanotechnology 2021, 16, 1251). For a fixed input miRNA concentration and charge ratio, NP diameters measure slightly smaller on average with more uniform size distributions utilizing FNC production (FIGs. 2D-2E). At these same conditions, the surface charge of the NPs, as reflected by their measured zeta potential (FIG. 2F), and the amount of miRNA encapsulated within them (FIG. 2G) were insensitive to mixing methodology. This insensitivity is reflective of the connection amongst zeta potential with charge neutralization and PEC formation (i.e., the number of residual positive charges at the NP surface), where miRNA-peptide binding is likely not rate-limiting in NP assembly, as has been observed for other nucleic acid-based PEC systems (Hu et al., ACS Nano 2019, 13, 10161). Using FNC, NP size showed a linear dependence on miRNA concentration, where increased concentrations afforded larger particles, and this linear dependence was absent when particles were prepared by bulk mixing (FIGs. 16A-16B). When charge ratio was varied (from N / P = 3 to 10), both NP size and zeta potential remain relatively constant regardless of mixing methodology (FIGs. 17A-17B). This further supports that miRNA-peptide charge neutralization and binding are likely not rate-limiting steps in NP assembly. Additionally, FNC production substantially reduces batch-to-batch variability compared to bulk mixing (FIGs. 2H-2I), significantly improving reproducibility. When exposing NPs to competition-induced decomplexation by addition of increasing concentrations of poly anionic heparin sulfate, NPs formed by FNC showed slightly higher EC 50 values (FIG. 2J), suggestive of the formation of tighter complexes. Excitingly, NPs produced by FNC exhibit enhanced colloidal stability, where their aggregation rate is reduced four- fold compared to bulk mixing (FIG. 2K). However, despite this slower rate, the NPs still aggregate within 24 hours by FNC (FIG. 2L). Aggregation is expedited by increasing input miRNA concentrations even moderately.

[0237] Enhancing Nanoparticle Stability through Peptide Design

[0238] We studied the effects of peptide primary charge and the manner in which charge is distributed along the peptide backbone to produce colloidally stable NPs. To achieve this, we explored a series of MAX 1 -derived peptide designs that vary both in the number and distribution of positively charged lysine residues (Figure 3 A). The sequence of MAXI contains two lysine-rich peptide strands that are connected to a central D-proline-L-Proline motif. When dissolved in water, MAXI is largely disordered. The two strand regions are random coil, but the diproline motif adopts distinct conformations that extend the two strands away from each other to limit charge repulsion. Each strand contains 4 lysines separated by hydrophobic valine residues. This design presents evenly distributed charge symmetrically about the central diproline motif. Peptide SSP1 is an isomer of MAXI that contains the same number lysine side-chains but displays them asymmetrically with respect to the diproline unit. This investigates how charge distribution affects particle assembly and stability (Figure 10). The TSS1 peptide contains two diproline motifs and an additional strand of VK repeats. This increases the overall peptide charge (+13) while evenly distributing the charge along the extended peptide (Figure 11). Peptide 0KM1 is similar to MAXI, but contains a four-residue repeat of lysines at its N-terminus, resulting in a charge of comparable to TSS1 (+13). Here, the charge is unevenly distributed along the peptide chain, containing a high density of charge at its terminus (Figure 12). Finally, the length of the poly-lysine tail was further increased, resulting in the PKM1 peptide, characterized by an overall charge of +17 (Figure 13). PKM1 tests the upper threshold for the number of charges that can be imbibed to the system.

[0239] First, the potential of each peptide to bind miRNA was assessed at neutral pH in water. We observe cooperative binding of all peptides to miRNA, where binding affinity increased with increasing number of lysines in the peptide design (isotherms in Figure 3B, 18). MAXI and SSP1, carrying the least charge, exhibited similar EC 50 values (100 vs 96 nM), suggesting for these peptides there is minimal influence of charge distribution. However, for the moderately charged peptides, 0KM1 exhibited a lower EC 50 value compared to TSS1 (78 vs 66 nM, both +13 charge), suggesting that charge distribution is important and that concentrating charge aids in miRNA binding. In the case of the most charged peptide PKM1, further increasing the charge density at the N-terminus decreased the EC 50 value to 50 nM. Interestingly, the EC50 of PKM1 is half that of MAXI , which contains half the number of lysine residues. Next, we determined the minimum charge ratio (N / P) necessary to complex a fixed concentration of miRNA across all the peptide designs. MAXI, SSP1, and TSS1 require a minimum N / P ratio of 3 to fully encapsulate the miRNA, whereas 0KM1 and PKM1 require a lower N / P ratio of 2 (Figure 3C-3G, respectively). This trend is mirrored in the EC 50 values in Figure 3B. Interestingly, although TSS1 and 0KM1 carry the same charge (+13), more copies of TSS1 are necessary to fully complex available miRNA, which suggests that the lysine-rich N-terminal tail of 0KM1 facilitates avid binding. For all peptides complexed with miRNA at an N / P ratio of 10, they are unfolded, adopting an intrinsically disordered state within their respective NPs (Figure 19), which is believed to be important for cellular entry and trafficking. Moreover, we compared the relative avidity of each miRNA-peptide complex by competition-induced decomplexation (Figure 20). This revealed a significant difference in avidity between MAXI and SSP1 (by an order of magnitude in EC50 value), despite the same number of charges, which may suggest a steric preference toward the shorter strand of SSP1 in binding to miRNA (i.e., less charge per peptide actively participating in miRNA-binding, hence the lower EC so). With the higher charged peptides, we observed a drop in ECso for heparin-induced decomplexation, which reflects a lower number of peptide molecules bound to a single miRNA and / or less charge per peptide participating in active binding to miRNA molecules.

[0240] To compare physiochemical properties of NPs formed by the different peptide designs, we first needed to determine the optimal processing parameters to achieve FNC production. We found our previously employed flow rate (Q = 20 mL min1) for MAXl-based NPs to be sufficient for achieving kinetic control of assembly of SSP1-, TSS 1-, and OKMl-based NPs with scramble miRNA (full characterization of the influence of IM on miRNA-TSS 1 NP size and uniformity in Figure 21). However, these same parameters proved unsuccessful for formulating miRNA-PKMl NPs. Interestingly, for the tested input miRNA concentration range (2 to 50 pg mL1) at a fixed charge ratio (N / P = 10), FNC failed to produce particles. NP formation was only possible using bulk mixing and high miRNA concentrations (above 20 pg mL1, Figure 22). However, lowering the charge ratio to N / P = 3, enabled nanoparticle formation by FNC (Q = 20 mL min1), while only slightly reducing complex avidity (Figure 23). The effect of inlet flow rate and thus mixing time on miRNA-PKMl NP size, uniformity, and DLS correlation function intercepts is shown in Figure 3H and 31. We again observe the existence of two regions of mixing kinetics, but in this case, the regions are centered around a single, optimal mixing time we term, TM.optimai (~ 40 ms, Q = 6 mL min1). To the best of our knowledge, this V-shaped plot of Figure 3H, with its singularity in optimal mixing time, is unique and has not yet been observed, highlighting the advantage of FNC in achieving exquisite kinetic control in formulating particles. At this flow rate (Q = 6 mL min1), miRNA-PKMl NPs can be produced with essentially zero batch-to-batch variability and a high degree of uniformity (Figure 25, 26).

[0241] Again, when the mixing time is greater than TM.optimai, the molecular events responsible for PEC chain formation, condensation, and association are diffusion-limited and occur nearly together, leading to larger and less uniform particles (Figure 24). Interestingly, when the mixing time is less than TM.optimai, nanoparticle formation is inhibited for the PKM1 system. The molecular mechanism defining particle formation is arrested after PEC chain formation (Figure 1C). In this time regime, mixing is rapid and not diffusion-limited. Thus, the binding of the highly charged peptide to miRNA most likely fully compensates its charge and leads to the formation of positively charged PEC chains unable to further self-associate. DLS confirms the inhibition of particle formation (Figure 24). Thus, the singularity in optimal mixing times seems to be dictated by peptide charge, and the +17 charge carried by PKM1 mostly likely defines the upper limit for allowing PEC chain association leading to NP formation.

[0242] Nanoparticles were then prepared using each peptide and their associated optimal FNC conditions to compare their physiochemical properties. Nanoparticles prepared with the more positively charged peptides exhibited smaller diameters and increased zeta potentials compared to MAXl-based NPs (Figure 3J, 3K). We assessed colloidal stability by monitoring particle aggregation over the course of 7 d in water through DLS size measurements. All newly designed peptides yielded NPs with remarkably improved stability compared to MAXl-based NPs (Figure 3L). We observe a direct correlation between zeta potential and colloidal stability, where miRNA- PKMl NPs with the highest zeta potential (+41 mV on average) showed no aggregation over the 7 d period (Figure 3M). In contrast and although exhibiting good stability, miRNA-TSSl NPs (+35 mV) showed a small initial size increase after 24 h that stabilized over rest of the experiment (Figure 3M).

[0243] Correlating peptide design to particle behavior is informative. It is clear that increasing the positive charge of the peptide directly correlates to increased values of zeta potential. However, zeta potentials do not strictly correlate with particle stability, suggesting that although increased charge is important, the manner in which that charge is displayed from the peptide backbone is crucial. For example, the sequences of MAXI and SSP1 carry the same formal charge and afford similarly charged particles, yet their particles exhibit vastly different stabilities (Figure 3K, 3L). The differential display of charge results in differential modes of miRNA binding (vide supra) and particle sizes, which most likely leads to differences in how residual charge is displayed at the surface of the particles, and thus affects their stability. Another case in point are the particles formed by TSS 1 and OKM 1 , peptides that both carry a formal charge of + 13. Their respective particles have similar zeta potentials (+35 vs +31 mV), yet TSS1 particles exhibit greater stability (Figure 3K, 3M). Ultimately, our exploration of peptide design yielded PKM1, whose unique display of charge affords particles that are stable for at least 3 months in water at room temperature (Figure 27), representing our most stable candidate.

[0244] EXAMPLE 3: TRANSFECTION EFFICIENCY AND GENE SILENCING EFFICACY OF PEC NPs IN VITRO

[0245] With successful formulation by FNC of the newly designed miRNA-peptide PEC NPs that exhibit enhanced colloidal stability, the biological activity of the NPs formulated with the different peptides (for PKM1, N / P = 3; otherwise, N / P = 10) was further assessed to characterize the effects of peptide design on their in vitro efficacy. To determine the transfection efficiency of each miRNA-peptide NP system, PEC NPs first were formulated with FAM-labeled scrambled miRNA and each different peptide and subsequently used to monitor cellular entry into MB52 mesothelioma cells by flow cytometry. All miRNA-peptide NPs tested are capable of successfully transfecting MB52 cells, but the transfection efficiency of MAXI- and PKMl-based NPs is very high and comparable to commercial transfection agent lipofectamine™ RNAiMAX (FIGs. 4A-4B). Previously, it has been reported that miRNA-215 functions as a tumor suppressor in mesothelioma by silencing the expression of MAD2L1, CDC7, LMNB2, and other genes which are critical for cell cycle regulation and proliferation (Singh et al., Molecular Therapy 2019, 27, 1665). Therefore, the antitumor efficacy of these PEC NPs using mesothelioma-specific tumor suppressor miRNA-215 as therapeutic cargo was evaluated. In MB52 cells, NPs formulated with miRNA-215 mimic and MAXI or PKM1 significantly decreased cell viability compared to respective scrambled miRNA- based NPs, and their efficacy was comparable to the lipofectamine control, while TSS1- and OKM 1 -based NPs elicited minimal decreases in cell viability (FIG. 4C, 28). The transfection efficiency and cytotoxicity trends observed here were also similar for treatment of another mesothelioma cell line (H2052), though more variability was observed (FIGs. 29A-29B). Furthermore, by measuring target gene expression levels, the gene silencing efficacy of each NP was evaluated using miRNA-215 as cargo in MB52 mesothelioma cells. Both miR-215-MAXl and miR-215-PKMl NPs significantly silenced the expression of MAD2L1 , CDC7, and LMNB2 genes compared to respective scrambled-miRNA NPs, while the NPs of other peptide designs did not show significant decrease in the expression of these critical genes (FIGs. 4D-4F, 30). Like cell viability, the silencing efficacy of miR-215-MAXl and miR-215-PKMl NPs silencing efficacy was comparable to the lipof ectamine control. Taken together, these data suggest that miRNA-215 -based PEC NPs formulated with either MAXI or PKM 1 peptides efficiently transfect mesothelioma cells and exert the biological activity of their specific miRNA cargo. In the context of PKM 1 -based NPs, their enhanced colloidal stability over MAXI -based NPs did not come at a cost of biological activity.

[0246] EXAMPLE 4: ENCAPSULATION OF PEC NPS INTO A FUNCTIONAL HYDROGEL

[0247] With lead NP designs delineated from both our stability and cell studies, we proceeded with formulating a hydrogel for their encapsulation. To accomplish this, an ice-chilled solution of a positively-charged hydrogelating peptide (HLT2, FIGs. 14A-14B) was mixed with a suspension of miRNA-peptide NPs followed by the addition of a saline buffer and heating this mixture to 37 °C. Salt-screening and increased temperature facilitates the self-assembly of the HLT2 peptide into a fibrillar gel that encapsulates the NPs (FIG. 5A). HLT2 was previously selected for the surface-fill hydrogel design, as its positive charge at physiological pH establishes repulsive forces between its fibrils and the NP cargo to facilitate their release from the gel network. NPs composed of FAM- labeled scramble miRNA complexed with MAXI, TSS1, or PKM1 were loaded into HLT2 gels and monitored for release from the gel network over 1 month in vitro (FIG. 5B). All NPs release from the hydrogel at a similar steady rate (around 60% of the payload released in 30 d) despite their differences in NP size and surface charge; however, this rate can be slightly modulated by varying the amount of HLT2 used to formulate the gel (FIG. 31). Oscillatory rheology shows that incorporation of the NPs had little influence on the bulk mechanical rigidity and the shear-thinning and recovery behavior of the SFH (empty SFH as a control, FIG. 5C-5H, 32). Further all SFH systems tested exhibited similar flow strains (FIGs. 5H, 33A-33H). Collectively, these experiments indicate that the gel-encapsulated particles can be delivered by syringe or spray for in vivo applications. EXAMPLE 5: TUMOR UPTAKE AND ANTITUMOR EFFICACY OF NP-CONTAINING HYDROGEL IN VIVO

[0248] With the new SFH formulations, tumor uptake and biodistribution of released miRNA- peptide PEC NPs were next evaluated in an in vivo xenograft model of mesothelioma. For the in vivo studies, higher concentrations of miRNA are used in the particle formulation to favor biological activity; however, the stability of the particles is unchanged (Figure 34). Briefly, gels containing Cyanine 3(Cy3)-labeled scrambled miRNA-MAXl or -PKM1 NPs were injected peritumorally to NOD.Cg-PrkdcscldII2rgt"",wNN / ] (NSG) mice bearing subcutaneous luciferaseexpressing MB52 mesothelioma tumors at 4 weeks post-cell inoculation (FIG. 6A). Establishment of tumors and peritumoral injection of gels were monitored at day 0 and day 4 post-implantation by monitoring both the tumor luminescence and Cy3 fluorescence in live imaging of mice (FIGs. 6 A, 35A-35B). For further analysis of the miRNA biodistribution, tumors and vital organs were collected from mice on day 4 of post-SFH administration for each treatment group (empty SFH control, Cy3-miR-MAXl NP-loaded SFH, and Cy3-miR-PKMl NP-loaded SFH) and imaged. From these images, Cy3 fluorescence signal revealed that Cy3-miR-MAXl and Cy3-miR-PKMl NPs release from the gel and enter tumor tissues as confirmed by tumor luminescence. No signal was observed in any vital organs such as liver, heart, kidney, spleen, lung and intestine, emphasizing the advantage of localized delivery (FIG. 6B). The observed absence of Cy3 fluorescence signal from tumors and vital organs collected from mice receiving empty gels rules out the possibility of tumor tissue autofluorescence (FIG. 6B). Furthermore, delivery of NPs to tumor cells was further assessed by histofluorescence imaging of tumor cross-sections taken from resected subcutaneous MB52 xenografts at day 4. Intracellular Cy3 fluorescence was observed in tumor tissue cross-sections from mice of both Cy3-miR-MAXl SFH and Cy3-miR-PKMl SFH treatment groups but not from the empty SFH group (FIG. 6C). These data suggest that the SFH locally delivers both miRNA-MAXl and miRNA-PKMl NPs to tumor cells with PKM1 particles qualitatively providing greater efficacy when injected peritumorally into mice bearing mesothelioma xenografts with no detectable delivery to other vital organs.

[0249] Next, we evaluated the antitumor efficacy of the miRNA-215-5p (miR-215)-carrying particles in the same xenograft model. Subcutaneous mesothelioma xenografts were established in NSG mice using luciferase-expressing MB 52 cells (FIG. 7A). After 4 weeks post-inoculation when tumors became established (volume had reached over 100 mm3), mice were randomly divided into four groups. Each treatment group was injected peritumorally with different SFH formulations (scr-miR-MAXl, miR-215-MAXl, scr-miR-PKMl, miR-215-PKMl ; scr-miR = scrambled miRNA). Live tumor luminescence imaging and tumor volume analysis at indicated time points over 30 days demonstrated that a single injection of SFH containing either miR-215-MAXl or miR-215-PKMl NPs significantly suppresses the growth of tumors in mice compared to their respective control group (FIGs. 7B-7D, 36). Additionally, tumor uptake of functional miRNA-215 NPs was analyzed in mesothelioma xenografts by qRT-PCR, which showed that miRNA-215 levels were significantly higher in tumor tissue resected from mice receiving a single peritumoral injection of SFH containing miR-215-PKMl NPs compared to mice receiving scr-miR-PKMl NPs at week 1 post-administration (FIG. 7E). The presence of miRNA-215 resulted in the silencing of target genes, MADL1, CDC7, and LMNB2 (FIGs. 7F-7H, respectively). Taken together, these data suggest that SFH containing the PKM1 -based NP can effectively deliver functional miRNA to tumor cells in vivo and exhibit antitumor activity with a single application. Further, at the higher miRNA concentrations necessary for the mice studies, MAXl-based NPs aggregate rapidly, whereas the PKM1 -based particles exhibit excellent colloidal stability, maintaining their average size and uniformity for at least 7 days in water at room temperature (FIGs. 38A-38D).

[0250] EXAMPLE 6: FORMULATING PEC NPs FOR OFF-THE-SHELF, FREEZE-DRIED STORAGE

[0251] While FNC production of the miRNA-peptide NPs is a highly scalable and reproducible process, their ability to be stored long-term would be a beneficial attribute. For example, NPs might be loaded into the gel on-site by clinicians just prior to use. Accordingly, freeze-drying of the nanoparticles was assessed as a long-term storage option. Freeze-dried scrambled miRNA- PKM1 particles that were reconstituted in water exhibited limited aggregation, and further it was found that an added cryoprotectant greatly influenced storage stability, protecting the freeze-dried particles against mechanical stresses present during freezing and reconstitution (Figure 8A, 8B). From the small panel of tested cryoprotectants (sucrose, trehalose, and D-mannitol), we found that addition of 2.5% w / v D-mannitol to be most effective at maintaining both NP size and polydispersity following lyophilization and reconstitution. The reconstitution process of lyophilized miRNA-PKMl NPs is very simple, where a clear NP suspension is generated after addition of water with minimal agitation at room temperature (Figure 8C). We next assessed maintenance of NP physiochemical properties over longer-term storage periods. We find that both miRNA-PKMl and miRNA-MAXl NPs are stable over a 3-month period of storage at -80°C (Figure 8D-8G). Although a slight increase in polydispersity was observed at 3 months for miRNA- PKMl NPs (Figure 8E), their size (Figure 8D), zeta potential (Figure 8F), miRNA loading (Figure 8G) are uncompromised. Freeze-drying did not affect the biological activity of the particles. NPs that had been stored for 1 month at 4°C or -80°C showed similar biological activity to freshly prepared particles. With respect to miRNA delivery, particles stored at -80°C performed a little better than those stored at 4°C (Figure 8H). Analysis of miRNA-215-5p (miR-215) target gene expression, MAD2L1, CDC7, and LMNB2 in MB52 cells revealed that the gene silencing efficacy of miR-215-PKMl NPs prepared freshly or lyophilized (stored at -80°C) is equivalent (Figure 81). Collectively, the data show that storage at -80°C to be ideal for preserving biological activity of these NPs. Lastly, the incorporation of either freeze-dried miRNA-PKMl or miRNA-MAXl NPs into gels had minimal influence on their bulk mechanical properties (Figure 39, 40). In fact, a slight increase in gel rigidity was observed in the presence of D-mannitol, which may be due to interactions between cryoprotectant and the hydrogel fibril network (Figure 39). Collectively, these results further emphasize the suitability of the freeze-drying methodology for miRNA-peptide NP formulation for loading into injectable hydrogels, demonstrating an avenue for SFH preparation that could be implemented in a clinical setting.

[0252] In view of the many possible aspects to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated aspects are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

It is claimed:

1. An isolated peptide comprising or consisting of an amino acid sequence set forth as: KKKKKKKKSGGVKVKVKVKVDPPTKVKVKVKV (PKM1, SEQ ID NO: 1) theDP is a proline that is a D amino acid; the C-terminus of the peptide is amidated or free carboxylic acid; the N-terminus of the peptide is acetylated or free amine; and the peptide is no more than 50 amino acids in length.

2. The isolated peptide of claim 1, wherein the peptide is from 35 to 50 amino acids in length.

3. The isolated peptide of claim 1 or claim 2, consisting of the amino acid sequence set forth asKKKKKKKKSGGVKVKVKVKVDPPTKVKVKVKV (PKM1, SEQ ID NO: 1)4. The isolated peptide of any one of claims 1-3, wherein the C-terminus of the peptide is amidated and the N-terminus of the peptide is acetylated.

5. The isolated peptide of any one of the prior claims, wherein the peptide forms an amphiphilic P-hairpin conformation in an aqueous solution comprising 150 mM NaCl and a pH of 7.4 at 25-37 °C.

6. A peptide hydrogel comprising nanoparticles comprising the isolated peptide of any one of claims 1-5.

7. A syringe containing the peptide hydrogel of claim 6.

8. A peptide hydrogel, comprising: nanoparticles encapsulated within the peptide hydrogel, wherein the nanoparticles comprise a nucleic acid molecule complexed with a first amphiphilic cationic P-hairpin peptide that is unfolded and not in a P-hairpin conformation, and wherein: the peptide hydrogel is formed from a fibrillar network of a second amphiphilic cationic - hairpin peptide that is in a -hairpin conformation;the first amphiphilic cationic -hairpin peptide is the isolated peptide of any one of claims1-5; the second amphiphilic cationic P-hairpin peptide comprises or consists of an amino acid sequence set forth as: VLTKVKTKVDPPTKVEVKVLV (HLT2, SEQ ID NO: 2), wherein theDP is a proline that is a D amino acid, the C-terminus of the second peptide is amidated or free carboxylic acid, the N-terminus of the second peptide is acetylated or free amine, and the second peptide is no more than 50 amino acids in length.

9. The peptide hydrogel of claim 8, wherein the second amphiphilic cationic P-hairpin peptide is from 25 to 50 amino acids in length.

10. The peptide hydrogel of claim 8 or claim 9, wherein the second amphiphilic cationic p-hairpin peptide consists of the amino acid sequence set forth as VLTKVKTKVDPPTKVEVKVLV (HLT2, SEQ ID NO: 2)11. The peptide hydrogel of any one of claims 8-10, wherein the C-terminus of the second amphiphilic cationic P-hairpin peptide is amidated and the N-terminus of the peptide is acetylated.

12. The peptide hydrogel of claim 8, wherein: the first amphiphilic cationic -hairpin peptide consists of the amino acid sequence set forth as KKKKKKKKSGGVKVKVKVKVDPPTKVKVKVKV (PKM1, SEQ ID NO: 1) and the C- terminus of the peptide is amidated and the N-terminus of the peptide is acetylated; the second amphiphilic cationic P-hairpin peptide consists of the amino acid sequence set forth as VLTKVKTKVDPPTKVEVKVLV (HLT2, SEQ ID NO: 2) the C-terminus of the peptide is amidated and the N-terminus of the peptide is acetylated.

13. The peptide hydrogel of any one of claims 8-12, wherein first amphiphilic cationic P-hairpin peptide forms an amphiphilic P-hairpin conformation in an aqueous solution comprising 150 mM NaCl and a pH of 7.4 at 25-37°C; and the second amphiphilic cationic P-hairpin peptide forms an amphiphilic P-hairpin conformation in an aqueous solution comprising 150 mM NaCl and a pH of 7.4 at 25-37°C.

14. The peptide hydrogel of any one of claims 8-13, wherein the nucleic acid molecule is an antisense nucleic acid molecule.

15. The peptide hydrogel of any one of claims 8-14, wherein the nucleic acid molecule is a microRNA or a mimic and / or mimetic thereof.

16. The peptide hydrogel of claim 15, wherein the nucleic acid molecule is any one of: hsa-miR-1 , hsa-miR-7, hsa-miR-lOa, hsa-miR-15, hsa-miR-16, hsa-miR-23a-3p, hsa-miR-24-1, hsa-miR-24-2-5p, hsa-miR-26a, hsa-miR-26b, hsa-miR-27a-5p, hsa-miR-27b-5p, hsa-miR- 27b-3p, hsa-miR-29a, hsa-miR-29b, hsa-miR-29c, hsa-miR-30b-5p, hsa-miR-30c-l, hsa-miR-33a, hsa-miR-34a, hsa-miR-95, hsa-miR-96, hsa-miR-100, hsa-miR-125a, hsa-miR-127, hsa-miR-130a, hsa-miR-130b, hsa-miR-132-3p, hsa-miR-133b, hsa-miR-134, hsa-miR-135a-l, hsa-miR-136, hsa- miR-139, hsa-miR-143, hsa-miR-145, hsa-miR-148a, hsa-miR-149, hsa-miR-181c, hsa-miR-181d, hsa-miR-182, hsa-mir-183, hsa-miR-190, hsa-miR-190b, hsa-miR-192, hsa-miR-195, hsa-miR-194, hsa-miR-200, hsa-miR-206, hsa-miR-212, hsa-miR-215, hsa-miR-221, hsa-miR-222-3p, hsa-miR- 320d-l, hsa-miR-342, hsa-miR-370, hsa-miR-375, hsa-miR-376a-l, hsa-miR-376b, hsa-miR-491, hsa-miR-497, hsa-miR-502, hsa-miR-506, hsa-miR-509-1, hsa-miR-548, hsa-miR-643, hsa-miR- 653, hsa-miR-664, hsa-miR-668, hsa-miR-676, hsa-miR-939, hsa-miR-1245, hsa-miR-1287, hsa- miR-1293, hsa-miR-1294, hsa-miR-1538, hsa-miR-2114, hsa-miR-3145, hsa-miR-3610, hsa-miR- 3677, hsa-let-7c-5p, hsa-miR-590-3p, hsa-miR-4472-1, hsa-miR-8078, hsa-miR-4675, hsa-mir-155, hsa-mir-196b, hsa-mir-4524a, or hsa-mir-4524b; or a mimic and / or mimetic thereof.

17. The peptide hydrogel of claim 16, wherein the nucleic acid molecule is any one of: miR-1, miR-145, miR-206, miR-215-5p, miR-491-5p, miR-497-5p, miR-1293, miR-634, miR- 5581-5p, miR-4498, miR-5681a, miR-3165, miR-342-5p, and miR-6715b-5p; or a mimic and / or mimetic thereof.

18. The peptide hydrogel of any one of claims 8-13, wherein the nucleic acid molecule is an mRNA.

19. The peptide hydrogel of any one of claims 8-13, wherein the nucleic acid molecule is a dinucleotide.

20. The peptide hydrogel of claim 19, wherein the dinucleotide is any one of 2'3'- cGAMP, c-di-GMP, or cAIMP.

21. The peptide hydrogel of any one of claims 8-20, further comprising a heterologous anti-cancer agent dispersed within the peptide hydrogel.

22. The peptide hydrogel of claim 21 , wherein the heterologous anti-cancer agent is a chemotherapeutic agent.

23. The peptide hydrogel of any one of claims 8-22, wherein the hydrogel undergoes shear-thinning upon application of shear stress, and rheological recovery upon removal of the shear stress.

24. The peptide hydrogel of any one of claims 8-23, comprising a storage modulus of greater than 40 Pascal in the absence of shear.

25. The peptide hydrogel of any one of claims 8-24, comprising from about 10 mM to about 400 mM NaCl and a pH of from about 7.0 to about 9.0.

26. The peptide hydrogel of any one of claims 8-25, comprising from about 0.25% to about 4.0 % w / v of the second amphiphilic cationic P-hairpin peptide.

27. The peptide hydrogel of claim 26, comprising from about 0.5% to about 2.0% w / v second amphiphilic cationic -hairpin peptide.

28. A syringe, containing the peptide hydrogel of any one of claims 8-27.

29. A method of administrating a nucleic acid molecule to a subject, comprising administering the peptide hydrogel of any one of claims 8-27 to a target location in the subject.

30. A method of treating or inhibiting a cancer in a subject, comprising administering an effective amount of the peptide hydrogel of any one of claims 8-27 to a target location in the subject where the cancer is present or is at risk of being present, wherein the nucleic acid molecule inhibits the cancer.

31. The method of claim 30, wherein the target location is a serosal surface lined by mesothelial cells in the subject where the cancer is present or is at risk of being present.

32. The method of claim 31 , wherein the serosal surface is part of a serosal body cavity in the subject.

33. The method of claim 32, wherein the serosal body cavity is a pleural cavity, a pericardial cavity, an anterior mediastinal cavity, a posterior mediastinal cavity, a peritoneal cavity, or a tunica vaginalis testis cavity.

34. The method of any one of claims 31-32, wherein administering the peptide hydrogel to the subject comprises injection or spray delivery of the peptide hydrogel to coat all or a portion of the serosal surface in the subject where the cancer is present or is at risk of being present.

35. The method of any one of claims 30-34, wherein the cancer is any one of a pleural mesothelioma, a peritoneal mesothelioma, a thymic epithelial cancer, an ovarian carcinoma, a cervical cancer, a small-cell lung carcinoma, a non-small-cell lung carcinoma, an ovarian carcinoma, an appendiceal cancer, a glioma, esophageal cancer, or a metastatic growth of a primary tumor that metastasized to a serosal surface in the subject.

36. The method of any one of claims 30-35, wherein the cancer is a serosal mesothelioma.

37. Use of the peptide hydrogel of any one of claims 8-27 to treat or inhibit a cancer in a subject, comprising administering an effective amount of the peptide hydrogel to a target location in the subject where the cancer is present or is at risk of being present, wherein the nucleic acid molecule inhibits the cancer.

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