In Vivo Mechanism of Action of Sodium Caprate for Improving the Intestinal Absorption of a GLP1/GIP Coagonist Peptide.

Sodium caprate (C10) has been widely evaluated as an intestinal permeation enhancer for the oral delivery of macromolecules. However, the effect of C10 on the intestinal absorption of peptides with different physicochemical properties and its permeation-enhancing effect in vivo remains to be understood. Here, we evaluated the effects of C10 on intestinal absorption in rats with a glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GIP-GLP1) dual agonist peptide (LY) and semaglutide with different enzymatic stabilities and self-association behaviors as well as the oral exposure of the LY peptide in minipigs. Furthermore, we investigated the mechanism of action (MoA) of C10 for improving the intestinal absorption of the LY peptide in vivo via live imaging of the rat intestinal epithelium and tissue distribution of the LY peptide in minipigs. The LY peptide showed higher proteolytic stability in pancreatin and was a monomer in solution compared to that in semaglutide. C10 increased in vitro permeability in the minipig intestinal organoid monolayer to a greater extent for the LY peptide than for semaglutide. In the rat jejunal closed-loop model, C10 increased the absorption of LY peptide better than that of semaglutide, which might be attributed to higher in vitro proteolytic stability and permeability of the LY peptide. Using confocal live imaging, we observed that C10 enabled the rapid oral absorption of a model macromolecule (FD4) in the rat intestine. In the duodenum tissues of minipigs, C10 was found to qualitatively reduce the tight junction protein level and allow peptide uptake to the intestinal cells. C10 decreased the transition temperature of the artificial lipid membrane, indicating an increase in membrane fluidity, which is consistent with the above in vivo imaging results. These data indicated that the LY's favorable physicochemical properties combined with the effects of C10 on the intestinal mucosa resulted in an ∼2% relative bioavailability in minipigs.

Identification of a Multi-Component Formulation for Intestinal Delivery of a GLP-1/Glucagon Co-agonist Peptide.

PURPOSE: Oral delivery of therapeutic peptides has been challenging due to multiple physiological factors and physicochemical properties of peptides. We report a systematic approach to identify formulation compositions combining a permeation enhancer and a peptidase inhibitor that minimize proteolytic degradation and increase absorption of a peptide across the small intestine. METHODS: An acylated glucagon-like peptide-1/glucagon co-agonist peptide (4.5 kDa) was selected as a model peptide. Proteolytic stability of the peptide was investigated in rat and pig SIF. Effective PEs and multiple component formulations were identified in rats. Relative bioavailability of the peptide was determined in minipigs via intraduodenal administration (ID) of enteric capsules. RESULTS: The peptide degraded rapidly in the rat and pig SIF. Citric acid, SBTI, and SBTCI inhibited the enzymatic degradation. The peptide self-associated into trimers in solution, however, addition of PEs monomerized the peptide. C10 was the most effective PE among tested PEs (DPC, LC, rhamnolipid, C12-maltosides, and SNAC) to improve intestinal absorption of the peptide in the rat IJ-closed loop model. A combination of C10 and SBTI or SBTCI increased the peptide exposure 5-tenfold compared to the exposure with the PE alone in the rat IJ-cannulated model, and achieved 1.06 ± 0.76% bioavailability in minipigs relative to subcutaneous via ID administration using enteric capsules. CONCLUSION: We identified SBTI and C10 as an effective peptidase inhibitor and PE for intestinal absorption of the peptide. The combination of SBTI and C10 addressed the peptide physiochemical properties and provides a formulation strategy to achieve intestinal delivery of this peptide.

Bottom-Up Fabrication of Multilayer Enteric Devices for the Oral Delivery of Peptides.

PURPOSE: To develop a planar, asymmetric, micro-scale oral drug delivery vehicle by i) fabricating microdevice bodies with enteric materials, ii) efficiently and stably loading sensitive drug molecules, and iii) capping microdevices for controlled drug release. METHODS: Picoliter-volume inkjet printing was used to fabricate microdevices through additive manufacturing via drop-by-drop deposition of enteric polymer materials. Microdevice bodies with reservoirs are fabricated through deposition of an enteric polymer, Eudragit FS 30 D. A model API, insulin, was loaded into each microdevice and retained its stability during printing and release. Eudragit L 100 and/or S 100 were used to cap microdevices and control the kinetics of insulin release in simulated intestinal conditions. RESULTS: Microdevice morphologies and size can be tuned on the fly based on printing parameters to span from the microscale to the mesoscale. Insulin retained its stability throughout device fabrication and during in vitro release in simulated intestinal conditions. Insulin release kinetics, from burst release to no release, can be tailored by controlling the blend of the Eudragit capping material. CONCLUSION: This approach represents a uniquely scalable and flexible strategy for microdevice fabrication that overcomes limitations in loading sensitive biologics and in the tuneability of device geometries that are inherent to traditional microfabrication strategies.

Rhamnolipids Enhance in Vivo Oral Bioavailability of Poorly Absorbed Molecules.

PURPOSE: This report describes the effect of rhamnolipids (RLs) on the tight junctions (TJ) of the intestinal epithelium using the rat in-situ closed loop model. METHODS: We investigated the transport of 5 (6)-carboxyfluorescein (CF) and fluorescein isothiocyanate-labeled dextrans with average molecular weights of 4.4 and 10 kDa (FD-4 and FD-10) when co-administered with different concentrations of RLs. Lactate dehydrogenase (LDH) leakage assay and histopathological examination of treated intestinal loops were used to assess potential toxicity of RLs. Further, the effect of kaempferol on accelerating the resealing of the tight junctions in vivo was also investigated RESULTS: Data shows that administration of different RLs concentrations (1.0-5.0% v/v) increased CF absorption through rat intestine by 2.84- and 15.82-folds with RLs concentrations of 1.0% and 5.0% v/v, respectively. RLs exhibited size-dependent increase on FD-4 and FD-10 absorption. Dosing RLs at 1.0% v/v didn't cause a significant LDH leakage or histopathological changes to intestinal mucosa compared to higher concentrations, which showed a progressive damaging effect. Using kaempferol, a natural flavonoid that stimulates the assembly of the TJs, proved to enhance the recovery of barrier properties of the intestinal mucosa treated with high concentrations of RLs (2.5% and 5% v/v). CONCLUSIONS: These results collectively illustrate the ability of RLs to enhance oral bioavailability of different molecules across the intestinal epithelial membrane in a concentration- and time-dependent fashion.

Noninvasive Ablation of Prostate Cancer Spheroids Using Acoustically-Activated Nanodroplets.

We have developed acoustically activated nanodroplets (NDs) using an amphiphilic triblock copolymer, which self-assembles and encapsulates different perfluorocarbons including perfluoropentane (PFP) and perfluorohexane (PFH). Applying histotripsy pulses (i.e., short, high pressure, ultrasound pulses) to solutions of PFP- and PFH-NDs generated bubble clouds at a significantly reduced acoustic pressure compared to the cavitation pressure observed for histotripsy treatment alone. In this report, we summarize the results of combining histotripsy at low frequency (345 and 500 kHz) with PFP-NDs and PFH-NDs on the ablation of PC-3 and C4-2B prostate cancer cells. Using custom built histotripsy transducers coupled to a microscope and a high speed recording camera, we imaged the generation of a cavitation bubble cloud in response to different ultrasound regimes in solution and in tissue-mimicking gel phantoms. We quantified the associated ablation of individual cancer cells and 3D spheroids suspended in solution and embedded in tissue phantoms to compare the ablative capacity of PFP-NDs and PFH-NDs. Results show that histotripsy pulses at high acoustic pressure (26.2 MPa) ablated 80% of prostate cancer spheroids embedded in tissue-mimicking gel phantoms. In comparison, combining histotripsy pulses at a dramatically lower acoustic pressure (12.8 MPa) with PFP-NDs and PFH-NDs caused an ablation of 40% and 80% of the tumor spheroid volumes, respectively. These results show the potential of acoustically activated NDs as an image-guided ablative therapy for solid tumors and highlight the higher ablative capacity of PFH-NDs, which correlates with the boiling point of the encapsulated PFH and the stability of the formed bubble cloud.

Formulation of Acid-Sensitive Micelles for Delivery of Cabazitaxel into Prostate Cancer Cells.

We report the synthesis of an amphiphilic triblock copolymer composed of a hydrophilic poly(ethylene glycol) (PEG) block, a central poly(acrylic acid) (PAA) block, and a hydrophobic poly(methyl methacrylate) (PMMA) block using atom transfer radical polymerization technique. We examined the self-assembly of PEG-b-PAA-b-PMMA copolymers in aqueous solutions forming nanosized micelles and their ability to encapsulate hydrophobic guest molecules such as Nile Red (NR) dye and cabazitaxel (CTX, an anticancer drug). We used 2,2ß'-(propane-2,2-diylbis(oxy))-diethanamine to react with the carboxylic acid groups of the central PAA block forming acid-labile, shell cross-linked micelles (SCLM). We investigated the loading efficiency and release of different guest molecules from non-cross-linked micelles (NSCLM) and shell cross-linked micelles (SCLM) prepared by reacting 50% (SCLM-50) and 100% (SCLM-100) of the carboxylic acid groups in the PAA in physiologic (pH 7.4) and acidic (pH 5.0) buffer solutions as a function of time. We examined the uptake of NR-loaded NSCLM, SCLM-50, and SCLM-100 micelles into PC-3 and C4-2B prostate cancer cells and the effect of different micelle compositions on membrane fluidity of both cell lines. We also investigated the effect of CTX-loaded NSCLM, SCLM-50, and SCLM-100 micelles on the viability of PC-3 and C4-2B cancer cells compared to free CTX as a function of drug concentration. Results show that PEG-b-PAA-b-PMMA polymers form micelles at concentrations ≥11 µg/mL with an average size of 40-50 nm. CTX was encapsulated in PEG-b-PAA-b-PMMA micelles with 55% loading efficiency in NSCLM. In vitro release studies showed that 30% and 85% of the loaded CTX was released from SCLM-50 micelles in physiologic (pH 7.4) and acidic (pH 5.0) buffer solutions over 30 h, confirming micelles' sensitivity to solution pH. Results show uptake of NSCLM and SCLM into prostate cancer cells delivering their chemotherapeutic cargo, which triggered efficient cancer cell death. PEG-b-PAA-b-PMMA micelles were not hemolytic and did not cause platelet aggregation, which indicate their biocompatibility.

Organization of Endothelial Cells, Pericytes, and Astrocytes into a 3D Microfluidic in Vitro Model of the Blood-Brain Barrier.

The endothelial cells lining the capillaries supplying the brain with oxygen and nutrients form a formidable barrier known as the blood-brain barrier (BBB), which exhibits selective permeability to small drug molecules and virtually impermeable to macromolecular therapeutics. Current in vitro BBB models fail to replicate this restrictive behavior due to poor integration of the endothelial cells with supporting cells (pericytes and astrocytes) following the correct anatomical organization observed in vivo. We report the coculture of mouse brain microvascular endothelial cells (b.End3), pericytes, with/without C8-D1A astrocytes in layered microfluidic channels forming three-dimensional (3D) bi- and triculture models of the BBB. The live/dead assay indicated high viability of all cultured cells up to 21 days. Trans-endothelial electrical resistance (TEER) values confirmed the formation of intact monolayers after 3 days in culture and showed statistically higher values for the triculture model compared to the single and biculture models. Screening the permeability of [(14)C]-mannitol and [(14)C]-urea showed the ability of bi- and triculture models to discriminate between different markers based on their size. Further, permeability of [(14)C]-mannitol across the triculture model after 18 days in culture matched its reported permeability across the BBB in vivo. Mathematical calculations also showed that the radius of the tight junctions pores (R) in the triculture model is similar to the reported diameter of the BBB in vivo. Finally, both the bi- and triculture models exhibited functional expression of the P-glycoprotein efflux pump, which increased with the increase in the number of days in culture. These results collectively indicate that the triculture model is a robust in vitro model of the BBB.

"Smart" Nanoparticles Enhance the Cytoplasmic Delivery of Anti-RhoC Silencing RNA and Inhibit the Migration and Invasion of Aggressive Breast Cancer Cells.

Rho-GTPases are small GTP-binding proteins that contribute to the epithelial-to-mesenchymal transition by regulating several cellular processes including organization of the actin cytoskeleton, cell motility, transcription, and cell proliferation. Overexpression of RhoC-GTPases (RhoC) in breast cancer has been implicated in poor disease prognosis due to increased cancer cells invasion, migration, and motility, which warranted its consideration as a therapeutic target for inhibiting breast cancer metastasis. Using silencing RNA (siRNA) molecules to knockdown RhoC expression is a promising approach to inhibit breast cancer metastases. However, transforming anti-RhoC siRNA molecules into a viable therapy remains a challenge due to the lack of a biocompatible carrier that can selectively deliver the RNA cargo into breast cancer cells. We report the use of a degradable, pH-sensitive, ß-cyclodextrin (ßCD)-based polymeric carrier that condenses anti-RhoC siRNA forming "smart" particles. These smart anti-RhoC particles were efficiently internalized, successfully escaped the endosome, and delivered the RNA cargo into the cytoplasm of SUM149 and MDA-MB-231 breast cancer cells. Our results show that anti-RhoC particles used at a low N/P ratio of 2.5/1 suppressed RhoC protein levels by 100% and 90% in SUM149 and MDA-MB-231 cells, respectively. Further, anti-RhoC particles inhibited the invasion, motility, and migration of SUM149 and MDA-MB-231 cells by 40-47%, 57-60%, and 61.5-73%, respectively. Smart particles encapsulating the scrambled siRNA sequence did not affect RhoC protein expression or the invasion, motility, and migration of SUM149 and MDA-MB-231 cells, which indicate the biocompatibility of the polymeric carrier and selectivity of the observed RhoC knockdown. These results collectively indicate the therapeutic potential of smart anti-RhoC particles in arresting the metastatic spread of breast cancer cells.

Effect of rhamnolipids on permeability across Caco-2 cell monolayers.

PURPOSE: This report describes the effect of rhamnolipids (RLs), an amphiphilic biosurfactant produced by the bacterium Pseudomonas aeruginosa, on the integrity and permeability across Caco-2 cell monolayers. METHODS: We measured the trans-epithelial electrical resistance (TEER) and permeability of [(14)C]mannitol across Caco-2 cell monolayers upon incubation with 0.01-5.0% v/v RLs as a function of incubation time (30, 60, 90, and 120 min). We also studied the recovery of RL-treated Caco-2 cell monolayers upon incubation with Kaempferol, which is a natural flavonoid that promotes the assembly of the tight junctions. RESULTS: TEER of Caco-2 cell monolayers incubated with 0.01-5.0% v/v RLs solution dropped to 80-28% of that of untreated cells. Decline in TEER was associated with an increase in [(14)C]mannitol permeability as a function of RLs concentration and incubation time with Caco-2 cells. Incubation of RLs-treated Caco-2 cell monolayers with normal culture medium for 48 h did not restore barrier integrity. Whereas, incubation of a RLs-treated Caco-2 cells with culture medium containing Kaempferol for 24 h restored barrier function indicated by the higher TEER and lower [(14)C]mannitol permeability values. CONCLUSIONS: These results show the ability of RLs to modulate the integrity and permeability of Caco-2 cell monolayers in a concentration- and time-dependent fashion, which suggest their potential to function as a non-toxic permeation enhancer.

Synergistic combination of small molecule inhibitor and RNA interference against antiapoptotic Bcl-2 protein in head and neck cancer cells.

B-cell lymphoma 2 (Bcl-2) is an antiapoptotic protein that is overexpressed in head and neck squamous cell carcinomas, which has been implicated in development of radio- and chemoresistance. Small molecule inhibitors such as AT-101 (a BH3-mimetic drug) have been developed to inhibit the antiapoptotic activity of Bcl-2 proteins, which proved effective in restoring radio- and chemo-sensitivity in head and neck cancer cells. However, high doses of AT-101 are associated with gastrointestinal, hepatic, and fertility side effects, which prompted the search for other Bcl-2 inhibitors. Short interfering RNA (siRNA) proved to inhibit antiapoptotic Bcl-2 protein expression and trigger cancer cell death. However, transforming siRNA molecules into a viable therapy remains a challenge due to the lack of efficient and biocompatible carriers. We report the development of degradable star-shaped polymers that proved to condense anti-Bcl-2 siRNA into "smart" pH-sensitive and membrane-destabilizing particles that shuttle their cargo past the endosomal membrane and into the cytoplasm of head and neck cancer cells. Results show that "smart" anti-Bcl-2 particles reduced the mRNA and protein levels of antiapoptotic Bcl-2 protein in UM-SCC-17B cancer cells by 50-60% and 65-75%, respectively. Results also show that combining "smart" anti-Bcl-2 particles with the IC25 of AT-101 (inhibitory concentration responsible for killing 25% of the cells) synergistically inhibits cancer cell proliferation and increases cell apoptosis, which reduce the survival of UM-SCC-17B cancer cells compared to treatment with AT-101 alone. Results indicate the therapeutic benefit of combining siRNA-mediated knockdown of antiapoptotic Bcl-2 protein expression with low doses of AT-101 for inhibiting the growth of head and neck cancer cells.

Progress and limitations of oral peptide delivery as a potentially transformative therapy.

INTRODUCTION: Numerous formulation technologies have been developed to overcome challenges of oral peptide delivery. Understanding the advantages and limitations of each technology is important for the development of new delivery systems to enable oral delivery of peptides designed for parenteral administration. AREAS COVERED: This review summarizes key learnings from the use of permeation enhancers (PEs) for oral peptide delivery associated with solid dosage form optimization to maximize the PE effect. Furthermore, we will highlight the most recent emerging delivery strategies to improve oral peptide bioavailability such as nanoparticles, self-emulsifying drug delivery systems, gut shuttles, and ingestible devices. In addition, advantages and limitations of these technologies will be compared with the permeation enhancer technology. EXPERT OPINION: Despite the success of permeation enhancer technology in the FDA-approved oral peptide products, oral peptide delivery is still facing the immense challenge of low-to-single digit oral bioavailability. Optimization of drug product attributes such as dissolution kinetics is critical to improve permeation enhancer efficacy. The next frontiers to substantially increase oral bioavailability and transform injectable peptides to oral deliverables may be ingestible devices and ligand-mediated transport (gut shuttles). However, clinical studies are necessary to inform the safety and efficacy of these emerging technologies.

Characterization and impact of peptide physicochemical properties on oral and subcutaneous delivery.

Peptides, an emerging modality within the biopharmaceutical industry, are often delivered subcutaneously with evolving prospects on oral delivery. Barrier biology within the subcutis or gastrointestinal tract is a significant challenge in limiting absorption or otherwise disrupting peptide disposition. Aspects of peptide pharmacokinetic performance and ADME can be mitigated with careful molecular design that tailors for properties such as effective size, hydrophobicity, net charge, proteolytic stability, and albumin binding. In this review, we endeavor to highlight effective techniques in qualifying physicochemical properties of peptides and discuss advancements of in vitro models of subcutaneous and oral delivery. Additionally, we will delineate empirical findings around the relationship of these physicochemical properties and in vivo (animal or human) impact. We conclude that robust peptide characterization methods and in vitro techniques with demonstrated correlations to in vivo data are key routines to incorporate in the drug discovery and development to improve the probability of technical and commercial success of peptide therapeutics.

Recent advances in proteolytic stability for peptide, protein, and antibody drug discovery.

Introduction: To discover and develop a peptide, protein, or antibody into a drug requires overcoming multiple challenges to obtain desired properties. Proteolytic stability is one of the challenges and deserves a focused investigation.Areas covered: This review concentrates on improving proteolytic stability by engineering the amino acids around the cleavage sites of a liable peptide, protein, or antibody. Peptidases are discussed on three levels including all peptidases in databases, mixtures based on organ and tissue types, and individual peptidases. The technique to identify cleavage sites is spotlighted on mass spectrometry-based approaches such as MALDI-TOF and LC-MS. For sequence engineering, the replacements that have been commonly applied with a higher chance of success are highlighted at the beginning, while the rarely used and more complicated replacements are discussed later. Although a one-size-fits-all approach does not exist to apply to different projects, this review provides a 3-step strategy for effectively and efficiently conducting the proteolytic stability experiments to achieve the eventual goal of improving the stability by engineering the molecule itself.Expert opinion: Improving the proteolytic stability is a spiraling up process sequenced by testing and engineering. There are many ways to engineer amino acids, but the choice must consider the cost and properties affected by the changes of the amino acids.

Effect of N-acetylgalactosamine ligand valency on targeting dendrimers to hepatic cancer cells.

The display of N-acetylgalactosamine (NAcGal) ligands has shown great potential in improving the targeting of various therapeutic molecules to hepatocellular carcinoma (HCC), a severe disease whose clinical treatment is severely hindered by limitations in delivery of therapeutic cargo. We previously used the display of NAcGal on generation 5 (G5) polyamidoamine (PAMAM) dendrimers connected through a poly(ethylene glycol) (PEG) brush (i.e. G5-cPEG-NAcGal; monoGal) to effectively target hepatic cancer cells and deliver a loaded therapeutic cargo. In this study, we were interested to see if tri-valent NAcGal ligands (i.e. NAcGal3) displayed on G5 dendrimers (i.e. G5-cPEG-NAcGal3; triGal) could improve their ability to target hepatic cancer cells compared to their monoGal counterparts. We therefore synthesized a library of triGal particles, with either 2, 4, 6, 8, 11, or 14 targeting branches (i.e. cPEG-NAcGal3) attached. Conventional flow cytometry studies showed that all particle formulations can label hepatic cancer cells in a concentration-dependent manner, reaching 90-100% of cells labeled at either 285 or 570 nM G5, but interestingly, monoGal labeled more cells at lower concentrations. To elucidate the difference in internalization of monoGal versus triGal conjugates, we turned to multi-spectral imaging flow cytometry and quantified the amount of internalized (I) versus surface-bound (I0) conjugates to determine the ratio of internalization (I/I0) in all treatment groups. Results show that regardless of NAcGal valency, or the density of targeting branches, all particles achieve full internalization and diffuse localization throughout the cell (I/I0 ∼ 3.0 for all particle compositions). This indicates that while tri-valent NAcGal is a promising technique for targeting nanoparticles to hepatic cancer cells, mono-valent NAcGal is more efficient, contrary to what is observed with small molecules.

Synergistic inhibition of aggressive breast cancer cell migration and invasion by cytoplasmic delivery of anti-RhoC silencing RNA and presentation of EPPT1 peptide on "smart" particles.

Overexpression of RhoC protein in breast cancer patients has been linked to increased cancer cell invasion, migration, and metastases. Suppressing RhoC expression in aggressive breast cancer cells using silencing RNA (siRNA) molecules is a viable strategy to inhibit the metastatic spread of breast cancer. In this report, we describe the synthesis of a series of asymmetric pH-sensitive, membrane-destabilizing polymers engineered to complex anti-RhoC siRNA molecules forming "smart" nanoparticles. Using ß-CD as the particle core, polyethylene glycol (PEG) chains were conjugated to the primary face via non-cleavable bonds and amphiphilic polymers incorporating hydrophobic and cationic monomers were grafted to the secondary face via acid-labile linkages. We investigated the effect of PEG molecular weight (2 & 5 kDa) on transfection capacity and serum stability of the formed particles. We evaluated the efficacy of EPPT1 peptides presented on the free tips of the PEG brush to function as a targeting ligand against underglycosylated MUC1 (uMUC1) receptors overexpressed on the surface of metastatic breast cancer cells. Results show that "smart" nanoparticles successfully delivered anti-RhoC siRNA into the cytoplasm of aggressive SUM149 and MDA-MB-231 breast cancer cells, which resulted in a dose-dependent inhibition of cell migration and invasion. Further, EPPT1-targeted nanoparticles demonstrate a synergistic inhibition of cell migration and invasion imparted via RhoC knockdown and EPPT1-mediated signaling via the uMUC1 receptor.

N-Acetylgalactosamine-Targeted Delivery of Dendrimer-Doxorubicin Conjugates Influences Doxorubicin Cytotoxicity and Metabolic Profile in Hepatic Cancer Cells.

This study describes the development of targeted, doxorubicin (DOX)-loaded generation 5 (G5) polyamidoamine dendrimers able to achieve cell-specific DOX delivery and release into the cytoplasm of hepatic cancer cells. G5 is functionalized with poly(ethylene glycol) (PEG) brushes displaying N-acetylgalactosamine (NAcGal) ligands to target hepatic cancer cells. DOX is attached to G5 through one of two aromatic azo-linkages, L3 or L4, achieving either P1 ((NAcGalß -PEGc)16.6 -G5-(L3-DOX)11.6 ) or P2 ((NAcGalß -PEGc)16.6 -G5-(L4-DOX)13.4 ) conjugates. After confirming the conjugates' biocompatibility, flow cytometry studies show P1/P2 achieve 100% uptake into hepatic cancer cells at 30-60 × 10-9 m particle concentration. This internalization correlates with cytotoxicity against HepG2 cells with 50% inhibitory concentration (IC50 ) values of 24.8, 1414.0, and 237.8 × 10-9 m for free DOX, P1, and P2, respectively. Differences in cytotoxicity prompted metabolomics analysis to identify the intracellular release behavior of DOX. Results show that P1/P2 release alternative DOX metabolites than free DOX. Stable isotope tracer studies show that the different metabolites induce different effects on metabolic cycles. Namely, free DOX reduces glycolysis and increases fatty acid oxidation, while P1/P2 increase glycolysis, likely as a response to high oxidative stress. Overall, P1/P2 conjugates offer a platform drug delivery technology for improving hepatic cancer therapy.

Dendrimer-doxorubicin conjugates exhibit improved anticancer activity and reduce doxorubicin-induced cardiotoxicity in a murine hepatocellular carcinoma model.

Hepatocellular carcinoma (HCC) is the 2nd leading cause of cancer-related deaths every year globally. The most common form of treatment, hepatic arterial infusion (HAI), involves the direct injection of doxorubicin (DOX) into the hepatic artery. It is plagued with limited therapeutic efficacy and the occurrence of severe toxicities (e.g. cardiotoxicity). We aim to improve the therapeutic index of DOX delivered via HAI by loading the drug onto generation 5 (G5) poly(amidoamine) (PAMAM) dendrimers targeted to hepatic cancer cells via N-acetylgalactosamine (NAcGal) ligands. DOX is attached to the surface of G5 molecules via two different enzyme-sensitive linkages, L3 or L4, to achieve controllable drug release inside hepatic cancer cells. We previously reported on P1 and P2 particles that resulted from the combination of NAcGal-targeting with L3- or L4-DOX linkages, respectively, and showed controllable DOX release and toxicity towards hepatic cancer cells comparable to free DOX. In this study, we demonstrate that while the intratumoral delivery of free DOX (1 mg/kg) into HCC-bearing nod scid gamma (NSG) mice achieves a 2.5-fold inhibition of tumor growth compared to the saline group over 30 days, P1 and P2 particles delivered at the same DOX dosage achieve a 5.1- and 4.4-fold inhibition, respectively. Incubation of the particles with human induced pluripotent stem cell derived cardiomyocytes (hiPSC CMs) showed no effect on monolayer viability, apoptosis induction, or CM electrophysiology, contrary to the effect of free DOX. Moreover, magnetic resonance imaging revealed that P1- and P2-treated mice maintained cardiac function after intraperitoneal administration of DOX at 1 mg/kg for 21 days, unlike the free DOX group at an equivalent dosage, confirming that P1/P2 can avoid DOX-induced cardiotoxicity. Taken together, these results highlight the ability of P1/P2 particles to improve the therapeutic index of DOX and offer a replacement therapy for clinical HCC treatment.

Effects of Droplet Composition on Nanodroplet-Mediated Histotripsy.

Nanodroplet-mediated histotripsy (NMH) is a targeted ablation technique combining histotripsy with nanodroplets that can be selectively delivered to tumor cells. In two previous studies, polymer-encapsulated perfluoropentane nanodroplets were used to generate well-defined ablation similar to that obtained with histotripsy, but at significantly lower pressure, when NMH therapy was applied at a pulse repetition frequency (PRF) of 10 Hz. However, cavitation was not maintained over multiple pulses when ultrasound was applied at a lower PRF (i.e., 1-5 Hz). We hypothesized that nanodroplets with a higher-boiling-point perfluorocarbon core would provide sustainable cavitation nuclei, allowing cavitation to be maintained over multiple pulses, even at low PRF, which is needed for efficient and complete tissue fractionation via histotripsy. To test this hypothesis, we investigated the effects of droplet composition on NMH therapy by applying histotripsy at various frequencies (345 kHz, 500 kHz, 1.5 MHz, 3 MHz) to tissue phantoms containing perfluoropentane (PFP, boiling point ∼29°C, surface tension ∼9.5 mN/m) and perfluorohexane (PFH, boiling point ∼56°C, surface tension ∼11.9 mN/m) nanodroplets. First, the effects of droplet composition on the NMH cavitation threshold were investigated, with results revealing a significant decrease (>10 MPa) in the peak negative pressure (p-) cavitation threshold for both types of nanodroplets compared with controls. A slight decrease (∼1-3 MPa) in threshold was observed for PFP phantoms compared with PFH phantoms. Next, the ability of nanodroplets to function as sustainable cavitation nuclei over multiple pulses was investigated, with results revealing that PFH nanodroplets were sustainable cavitation nuclei over 1,000 pulses, whereas PFP nanodroplets were destroyed during the first few pulses (<50 pulses), likely because of the lower boiling point. Finally, tissue phantoms containing a layer of embedded red blood cells were used to compare the damage generated for NMH treatments using PFP and PFH droplets, with results indicating that PFH nanodroplets significantly improved NMH ablation, allowing for well-defined lesions to be generated at all frequencies and PRFs tested. Overall, the results of this study provide significant insight into the role of droplet composition in NMH therapy and provide a rational basis to tailor droplet parameters to improve NMH tissue fractionation.

Silencing bcl-2 expression in epithelial cancer cells using "smart" particles.

Short interfering RNA (siRNA) targeted against anti-apoptotic Bcl-2 protein proved to knockdown its expression and trigger cancer cell death. We used degradable, pH-sensitive, comb-like [P(EAA-co-BMA)-b-PNASI-g-P(HMA-co-TMAEMA)] polymer to condense anti-Bcl-2 siRNA into "smart" particles, which proved to shuttle their cargo past the endosomal membrane and into the cytoplasm of HeLa and UM-SCC-17B cancer cells. HeLa and UM-SCC-17B cancer cells were treated with anti-Bcl-2 particles followed by quantifying Bcl-2 mRNA and protein levels using qRT-PCR and western blotting, respectively. "Smart" anti-Bcl-2 particles selectively suppress Bcl-2 mRNA and protein levels in HeLa cells by 50%-60% and 79%-81%, respectively. Similarly, "smart" anti-Bcl-2 particles inhibited Bcl-2 mRNA levels by 30%, 40%, and 20% upon incubation with UM-SCC-17B cancer cells for 48, 72, and 96 h, respectively. Bcl-2 protein expression in UM-SCC-17B cancer cells was inhibited by 30% after treatment for 72 h. Results show that pH-sensitive comb-like polymer complex anti-Bcl-2 siRNA forming "smart" nanoparticles that deliver their cargo into the cytoplasm of HeLa and UM-SCC-17B cancer cells causing Bcl-2 knockdown at the mRNA and protein levels.

Visualizing the attack of RNase enzymes on dendriplexes and naked RNA using atomic force microscopy.

Cationic polymers such as poly(amidoamine), PAMAM, dendrimers have been used to electrostatically complex siRNA molecules forming dendriplexes for enhancing the cytoplasmic delivery of the encapsulated cargo. However, excess PAMAM dendrimers is typically used to protect the loaded siRNA against enzymatic attack, which results in systemic toxicity that hinders the in vivo use of these particles. In this paper, we evaluate the ability of G4 (flexible) and G5 (rigid) dendrimers to complex model siRNA molecules at low +/- ratio of 2/1 upon incubation for 20 minutes and 24 hours. We examine the ability of the formed G4 and G5 dendriplexes to shield the loaded siRNA molecules and protect them from degradation by RNase V1 enzymes using atomic force microscopy (AFM). Results show that G4 and G5 dendrimers form similar hexagonal complexes upon incubation with siRNA molecules for 20 minutes with average full width of 43±19.3 nm and 62±8.3 at half the maximum height, respectively. AFM images show that these G4 and G5 dendriplexes were attacked by RNase V1 enzyme leading to degradation of the exposed RNA molecules that increased with the increase in incubation time. In comparison, incubating G4 and G5 dendrimers with siRNA for 24 hours led to the formation of large particles with average full width of 263±60 nm and 48.3±2.5 nm at half the maximum height, respectively. Both G4 and G5 dendriplexes had a dense central core that proved to shield the loaded RNA molecules from enzymatic attack for up to 60 minutes. These results show the feasibility of formulating G4 and G5 dendriplexes at a low N/P (+/-) ratio that can resist degradation by RNase enzymes, which reduces the risk of inducing non-specific toxicity when used in vivo.