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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.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.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.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.

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.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.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.

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 challenge...

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.

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.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).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.

WOS: 000373550600023 PubMed ID: 26977718 We report the synthesis of an amphiphilic triblock copolymer composed of a hydrophilic polyethylene 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 2,2 beta'-(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 mu g/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. National Ministry of Education of the Republic of Turkey [1416]; Ministry of Higher Education of the Arab Republic of Egypt O.A. wishes to recognize the financial support of the National Ministry of Education of the Republic of Turkey (Program #:1416). I.Y. recognizes the financial support of the Ministry of Higher Education of the Arab Republic of Egypt through the Joint Supervision Fellowship Program. I.Y. also recognizes the support of Dr. Ahmed Fadda, Dr. ElSayed Afsah, and Dr. Ibrahim El-Sherbiny for serving as the dissertation committee at Mansoura University, Mansoura, Egypt. The authors thank Dr. Daniel Eitzman and Dr. Jinsang Kim at the University of Michigan for granting access to the Aggro-Link Data Reduction System (Chrono-log Corporation, Havertown, PA) and the QM4 Fluorescence Spectrophotometer (PerkinElmer, Waltham, MA) used to measure platelets aggregation and changes in albumin fluorescence, respectively. The authors also thank Mr. Ahmet Emrehan Emre for his technical assistance with electron microscopy imaging.

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. 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. 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. 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.

WOS: 000361899900007 PubMed ID: 25959056 Nanodroplet-mediated histotripsy (NMH) is a targeted ultrasound ablation technique combining histotripsy with nanodroplets that can be selectively delivered to tumor cells for targeted tumor ablation. In a previous study, it was reported that by use of extremely short, high-pressure pulses, histotripsy cavitation bubbles were generated in regions containing nanodroplets at significantly lower pressure (similar to 10.8 MPa) than without nanodroplets (similar to 28 MPa) at 500 kHz. Furthermore, it was hypothesized that lower frequency would improve the effectiveness of NMH by increasing the size of the focal region, increasing bubble expansion, and decreasing the cavitation threshold. In this study, we investigated the effects of ultrasound frequency (345 kHz, 500 kHz, 1.5 MHz, and 3 MHz) on NMH. First, the NMH cavitation threshold was measured in tissue phantoms with and without nanodroplets, with results indicating that the NMH threshold was significantly below the histotripsy intrinsic threshold at all frequencies. Results also indicated that the NMH threshold decreased at lower frequency, ranging from 7.4 MPa at 345 kHz to 13.2 MPa at 3 MHz. In the second part of this study, the effects of frequency on NMH bubble expansion were investigated, with results indicating larger expansion at lower frequency, even at a lower pressure. In the final part of this study, the ability of perfluoropentane-encapsulated nanodroplets to act as sustainable cavitation nuclei over multiple pulses was investigated, with results indicating that the nanodroplets are destroyed by the cavitation process and only function as cavitation nuclei for the first few pulses, with this effect being most pronounced at higher frequencies. Overall, the results of this study support our hypothesis that using a lower frequency will improve the effectiveness of NMH by increasing the size of the focal region, increasing bubble expansion and decreasing the cavitation threshold. National Science Foundation Graduate Research Fellowship; Turkish Republic the Ministry of National Education Fellowship Program [1416]; U.S. Department of Defense [W81 XWH-11-PCRP-ID]

An entirely new approach to tissue engineering is presented that uses the interfacial forces between aqueous solutions of phase-separating polymers to confine cells and promote their assembly into interconnected, macroscopic tissue constructs. This simple and inexpensive general procedure creates free-standing, centimeter-scale constructs from cell suspensions at the interface between poly(ethylene glycol) and dextran aqueous two-phase systems in as little as 2 h. Using this method, skin constructs are produced that integrate with decellularized dermal matrices, on which they differentiate and stratify into skin equivalents. It is demonstrated that the constructs produced by this method have appropriate integrity and mechanical properties for use as in vitro tissue models.

Objectives: The aim of this study was to investigate the ability of siRNA-loaded nanoparticles to control the capability of cisplatin-resistant cell lines to decrease cancer metastasis and cell proliferation rate. Methods: The effect of siRNA-loaded NPs on the migratory (metastasis) and proliferation (cell growth rate) activity of cisplatin-resistant cell lines compared to untreated cells was investigated using the wound healing assay and cell growth rate assay, respectively. Results: The results showed that cells were treated with siRNA-loaded nanoparticles after 24 h from starting the scratch, succeeded to cover of 59 ± 12.9, and 30 ± 6.1% only from the wound for cell line Cis 1R and Cis 12R, respectively. On the other hand of the untreated cells that were treated with positive control nanoparticles showed complete healing for the wound within the same period of time. Results of cell proliferation of both cell lines exhibited significantly (P≤ 0.001) lower growth rate after treatment with loaded nanoparticles compared to positive control nanoparticles and untreated cells. Conclusion: The obtained results indicated that siRNA-loaded nanoparticles succeeded to not only reduce metastasis but also decrease cell proliferation rate of cisplatin-resistant cell lines.

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.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.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.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.

B-cell lymphoma 2 (Bcl-2) is an anti-apoptotic protein that is over-expressed in head and neck squamous cell carcinomas, which has been implicated in development of radio- and chemo-resistance. Small molecule inhibitors such as AT-101 (a BH3-mimetic drug) have been developed to inhibit the anti-apoptotic 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 anti-apoptotic 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 anti-apoptotic 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 inhibit cancer cell proliferation and increase cell apoptosis, which reduced 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 anti-apoptotic Bcl-2 protein expression with low doses of AT-101 for inhibiting the growth of head and neck cancer cells.

We report the synthesis of a series of aromatic azo-linkers (L1–L4), which are selectively recognized and cleaved by azoreductase enzymes present in the cytoplasm of hepatic cancer cells via a NADPH-dependent mechanism. We utilized L1–L4 azo-linkers to conjugate doxorubicin to generation 5 (G5) of poly(amidoamine) dendrimers to prepare G5–L( x )–DOX nanoconjugates. We incorporated electron-donating oxygen (O) or nitrogen (N) groups in the para and ortho positions of L1–L4 azo-linkers to control the electronegativity of G5–L( x )–DOX conjugates and investigated their cleavage by azoreductase enzymes and the associated release of loaded DOX molecules. Hammett σ values of G5–L( x )–DOX conjugates ranged from −0.44 to −1.27, which is below the reported σ threshold (−0.37) required for binding to azoreductase enzymes. Results show that incubation of G5–L1–DOX ( σ = −0.44), G5–L2–DOX ( σ = −0.71), G5–L3–DOX ( σ = −1.00), and G5–L4–DOX ( σ = −1.27) conjugates with human liver microsomal (HLM) enzymes and the S9 fraction isolated from HepG2 hepatic cancer cells results in release of 4%–8%, 17%, 60%, and 100% of the conjugated DOX molecules, respectively. These results show that increasing the electronegativity (i.e. lower σ value) of L1–L4 azo-linkers increases their susceptibility to cleavage by azoreductase enzymes. Intracellular cleavage of G5–L( x )–DOX nanoconjugates, release of conjugated DOX molecules, and cytotoxicity correlated with conjugate's electronegativity ( σ value) was investigated, with G5–L4–DOX conjugate exhibiting the highest toxicity towards hepatic cancer cells with an IC 50 of 13 n m ± 5 n m in HepG2 cells. Cleavage of G5–L( x )–DOX conjugates was specific to hepatic cancer cells as shown by low non-specific DOX release upon incubation with non-enzymatic insect proteins and the S9 fraction isolated from rat cardiomyocytes. These enzyme-activated G5–L( x )–DOX conjugates represent a drug delivery platform that can achieve tunable and cell-specific release of the loaded cargo in hepatic cancer cells.

The report describes the synthesis of degradable, pH-sensitive, membrane-destabilizing, star-shaped polymers where copolymers of hydrophobic hexyl methacrylate (HMA) and 2-(dimethylamino)ethyl methacrylate (DMAEMA) monomers are grafted from the secondary face of a beta-cyclodextrin (β-CD) core via acid-labile hydrazone linkages using atom transfer radical polymerization. The effect of the graft's molecular weight, HMA/DMAEMA molar ratio, and the fraction of DMAEMA converted to cationic N,N,N-trimethylaminoethyl methacrylate (TMAEMA) monomers on polymer's transfection capacity is systematically investigated. Results show that all star-shaped polymers condense anti-GAPDH silencing RNA (siRNA) into nanosized particles at +/- ratio ≤ 4:1. Star polymers with shorter (25kDa) P(HMA-co-DMAEMA-co-TMAEMA) grafts are more efficient and less cytotoxic than carriers with longer (40kDa) grafts. The results show that increasing the ratio of hydrophobic HMA monomers in graft's composition higher than 50 mole% dramatically reduces polymer's aqueous solubility and abolishes their transfection capacity. Further, retention of DMAEMA monomers in graft's composition provide a buffering capacity that enhanced the endosomal escape and transfection capacity of the polymers. These systematic studies show that β-CD-P(HMA-co-DMAEMA-co-TMAEMA)4.8 polymer with a 25 kDa average graft's molecular weight and a 50/25/25 ratio of HMA/DMAEMA/TMAEMA monomers is the most efficient carrier in delivering the siRNA cargo into the cytoplasm of epithelial cancer cells.

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.

This paper is an initial work towards developing an image-guided, targeted ultrasound ablation technique by combining histotripsy with nanodroplets that can be selectively delivered to tumor cells. Using extremely short, high-pressure pulses, histotripsy generates a dense cloud of cavitating microbubbles that fractionates tissue. We hypothesize that synthetic nanodroplets that encapsulate a perfluoropentane (PFP) core will transition upon exposure to ultrasound pulses into gas microbubbles, which will rapidly expand and collapse resulting in disruption of cells similar to the histotripsy process but at a significantly lower acoustic pressure. The significantly reduced cavitation threshold will allow histotripsy to be selectively delivered to the tumor tissue and greatly enhance the treatment efficiency while sparing neighboring healthy tissue. To test our hypothesis, we prepared nanodroplets with an average diameter of 204±4.7 nm at 37°C by self-assembly of an amphiphilic triblock copolymer around a PFP core followed by cross-linkage of the polymer shell forming stable nanodroplets. The nanodroplets were embedded in agarose tissue phantoms containing a sheet of red blood cells (RBCs), which were exposed to 2-cycle pulses applied by a 500 kHz focused transducer. Using a high speed camera to monitor microbubble generation, the peak negative pressure threshold needed to generate bubbles >50 μm in agarose phantoms containing nanodroplets was measured to be 10.8 MPa, which is significantly lower than the 28.8 MPa observed using ultrasound pulses alone. High speed images also showed cavitation microbubbles produced from the nanodroplets displayed expansion and collapse similar to histotripsy alone at higher pressures. Nanodroplet-mediated histotripsy created consistent, well-defined fractionation of the RBCs in agarose tissue phantoms at 10 Hz pulse repetition frequency similar to the lesions generated by histotripsy alone but at a significantly lower pressure. These results support our hypothesis and demonstrate the potential of using nanodroplet-mediated histotripsy for targeted cell ablation.

WOS: 000389497700004 PubMed ID: 27696857 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. Turkish Republic the Ministry of National Education PhD Fellowship Program [1416]; National Science Foundation Graduate Research Fellowship; United States Department of Defense [W81XVVH-11-PCRP-ID] We would like to thank S. Capracotta, PhD (Technical Specialist, Nano Sight, School of Public Health, University of Michigan), for her help on NTA size and concentration measurements. OA. acknowledges the support of the Turkish Republic the Ministry of National Education PhD Fellowship Program (1416). This material is based upon work supported by a National Science Foundation Graduate Research Fellowship to E.V. This work was supported by a grant from the United States Department of Defense (W81XVVH-11-PCRP-ID) to M.E.H.E.

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 ((NAcGalβ -PEGc)16.6 -G5-(L3-DOX)11.6 ) or P2 ((NAcGalβ -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.

WOS: 000373384700012 PubMed ID: 26774470 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, whenNMHtherapy 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 similar to 29 degrees C, surface tension similar to 9.5 mN/m) and perfluorohexane (PFH, boiling point similar to 56 degrees C, surface tension similar to 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 (similar to 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 (

Microfluidic devices fabricated using poly(dimethylsiloxane) (PDMS) polymer are routinely used for in vitro cell culture for a wide range of cellular assays. These assays typically involve the incubation of cultured cells with a drug molecule or a fluorescent marker while monitoring a cellular response. The accuracy of these assays depends on achieving a consistent and reproducible concentration of solute molecules in solution. However, hydrophobic therapeutic and fluorescent molecules tend to diffuse into the PDMS walls of the microfluidic devices, which reduce their concentration in solution and consequently affect the accuracy and reliability of these assays. In this paper, we quantitatively investigate the relationship between the partition coefficient (log P) of a series of markers routinely used in in vitro cellular assays including [3H]-dexamethasone, [3H]-diazepam, [14C]-mannitol, [3H]-phenytoin, and rhodamine 6G and their absorption into PDMS microfluidic channels. Our results show that the absorption of a given solute into PDMS depends on the hydrophilic/hydrophobic balance defined by its log P value. Specifically, results demonstrate that molecules with log P less than 2.47 exhibit minimal absorption (10%) into PDMS channels whereas molecules with log P larger than 2.62 exhibit extensive absorption (90%) into PDMS channels. Further investigations showed that TiO(2) and glass coatings of PDMS channels reduced the absorption of hydrophobic molecules (log P2.62) by 2- and 4.5-folds, respectively.

There is an urgent need for novel polymeric carriers that can selectively deliver a large dose of chemotherapeutic agents into hepatic cancer cells to achieve high therapeutic activity with minimal systemic side effects. PAMAM dendrimers are characterized by a unique branching architecture and a large number of chemical surface groups suitable for coupling of chemotherapeutic agents. In this article, we report the coupling of N-acetylgalactosamine (NAcGal) to generation 5 (G5) of poly(amidoamine) (PAMAM-NH₂) dendrimers via peptide and thiourea linkages to prepare NAcGal-targeted carriers used for targeted delivery of chemotherapeutic agents into hepatic cancer cells. We describe the uptake of NAcGal-targeted and non-targeted G5 dendrimers into hepatic cancer cells (HepG2) as a function of G5 concentration and incubation time. We examine the contribution of the asialoglycoprotein receptor (ASGPR) to the internalization of NAcGal-targeted dendrimers into hepatic cancer cells through a competitive inhibition assay. Our results show that uptake of NAcGal-targeted G5 dendrimers into hepatic cancer cells occurs via ASGPR-mediated endocytosis. Internalization of these targeted carriers increased with the increase in G5 concentration and incubation time following Michaelis-Menten kinetics characteristic of receptor-mediated endocytosis. These results collectively indicate that G5-NAcGal conjugates function as targeted carriers for selective delivery of chemotherapeutic agents into hepatic cancer cells.

Recent advances in drug design have led to the development of new small molecular weight chemotherapeutic agents, peptides, proteins, and nucleic acid molecules that can be used for treatment of breast cancer. However, transformation of these drug candidates into actual therapies with well-defined dosing regimens remains a significant challenge due to the limited ability to selectively deliver these drug molecules into the cytoplasm of breast cancer cells. In this article, we describe the use of liposomes, dendrimers, and polymeric micelles as carriers that can be used for delivery of anticancer drugs and their potential in breast cancer therapy. We also summarize the challenges facing the development of nucleic acid–based therapies.

The influence of solute hydrophobicity and charge on partitioning and diffusion in physically crosslinked networks of a genetically engineered SELP polymer was investigated. A series of fluorescent dyes were used to assess the impact of solute charge and hydrophobicity on release behavior. The mechanism of solute release from the SELP hydrogel appeared to vary as a function of dye hydrophobicity. The extent of FITC attachment to amine-terminated G4 dendrimers influenced SELP hydrogel partitioning more than dendrimer diffusion properties. Results suggest the possibility of controlling solute release from SELP hydrogels by modifying the hydrophobicity and surface charge of drugs and drug/polymer conjugates as well as the possibility of "designing-in" solute-specific interactions.

Localized delivery of therapeutic agents through the blood–brain barrier (BBB) is a clinically significant task that remains challenging. Ultrasound (US) application after intravenous administration of microbubbles has been shown to generate localized BBB opening in animal models but the detailed mechanisms are not yet fully described. The current study investigates the effects of US-stimulated microbubbles on in vitro murine brain microvascular endothelial (bEnd.3) cells by monitoring sonoporation and changes in intracellular calcium concentration ([Ca2+]i) using real-time fluorescence and high-speed brightfield microscopy. Cells seeded in microchannels were exposed to a single US pulse (1.25 MHz, 10 cycles, 0.24 MPa peak negative pressure) in the presence of Definity™ microbubbles and extracellular calcium concentration [Ca2+]o = 0.9 mM. Disruption of the cell membrane was assessed using propidium iodide (PI) and change in the [Ca2+]i was measured using fura-2. Cells adjacent to a microbubble exhibited immediate [Ca2+]i changes after US pulse with and without PI uptake and the [Ca2+]i changes were twice as large in cells with PI uptake. Cell viability assays showed that sonoporated cells could survive with modulation of [Ca2+]i and uptake of PI. Cells located near sonoporated cells were observed to exhibit changes in [Ca2+]i that were delayed from the time of US application and without PI uptake. These results demonstrate that US-stimulated microbubbles not only directly cause changes in [Ca2+]i in brain endothelial cells in addition to sonoporation but also generate [Ca2+]i transients in cells not directly interacting with microbubbles, thereby affecting cells in larger regions beyond the cells in contact with microbubbles. (E-mail: cxdeng@umich.edu )

This manuscript describes a straightforward fabrication process for embedding Ag/AgCl electrodes within a two-layer poly(dimethylsiloxane) (PDMS) microfluidic chip where an upper and a lower channel are separated by a semiporous membrane. This system allows for the reliable real-time measurement of transendothelial and transepithelial electrical resistance (TEER), an accepted quantification of cell monolayer integrity, across cells cultured on membranes inside the microchannels using impedance spectroscopy. The technique eliminates the need for costly or specialized microelectrode fabrication, enabling commercially available wire electrodes to easily be incorporated into PDMS microsystems for measuring TEER under microfluidic environments. The capability of measuring impedance across a confluent cell monolayer is confirmed using (i) brain-derived endothelial cells (bEND.3), (ii) Madin Darby Canine Kidney Cells (MDCK-2), and mouse myoblast (C2C12) (all from ATCC, Manassas, VA). TEER values as a function of cell type and cell culture time were measured and both agree with previously published values from macroscale culture techniques. This system opens new opportunities for conveniently resolving both transendothelial and transepithelial electrical resistance to monitor cell function in real-time in microfluidic cell cultures.

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.

Colonic epithelium composes of various cell types including alkaline phosphate-expressing absorptive, mucussecreting goblet and neuroendocrine cells that are derived from stem cells through asymmetric division. The continuous renewal of stem cells occurs under the highly coordinated cellular redox state. In the current study, based on a comparison with other culture media, colon epithelial cells were able to be sustained in vitro with normal status for more than two months under the chosen culture condition; α-MEM medium containing 20% fetal bovine serum. The cultured epithelial cells had normal doubling time and normal morphological characteristics as examined by transmission electron microscope. Also, these cultured cells contained functional stem cells and maintained their differentiation potency of colon stem cells, compared with freshly isolated mucosal epithelial cells, as indicated by the maintaining of aldehyde dehydrogenase 1B1 expression (11.31 ± 0.45 to 11.15 ± 0.48), ability to reduce silver nitrate, alkaline phosphate activity (0.513 ± 0.007 mU/μg to 0.438 ± 0.005 mU/μg), mucin secretion (34.71 ± 0.714 μg/ml to 32.93 ± 0.357 μg/ml) in appropriate cellular redox state level (-258.4 ± 1.3 mV to -237.4 ± 3.7 mV). The present study showed sustaining replication potential and functional differentiation of colonic epithelial stem cell population in this culture. The above culture system may be useful as an in vitro model for stemness, toxicological, and carcinogenesis studies.

Many macromolecular therapeutics such as peptides, proteins, antisense oligodeoxynucleotides (ASODN), and short interfering RNA (siRNA) are active only in the cytoplasm or nucleus of targeted cells. Endocytosis is the primary route for cellular uptake of these molecules, which results in their accumulation in the endosomal-lysosomal trafficking pathway and loss of therapeutic activity. In this article, we describe the synthesis and pH-dependent membrane-destabilizing activity of a new "smart" polymer family that can be utilized to enhance the intracellular delivery of therapeutic macromolecules through the endosomal membrane barrier into the cytoplasm of targeted cells. These polymers are propylamine, butylamine, and pentylamine derivatives of poly(styrene-alt-maleic anhydride) (PSMA) copolymers. The PSMA-alkylamide derivatives are hydrophilic and membrane-inactive at physiological pH; however, they become hydrophobic and membrane-disruptive in response to endosomal pH values as measured by their hemolytic activity. Results show that the pH-dependent membrane-destabilizing activity of PSMA derivatives can be controlled by varying the length of the alkylamine group, the degree of modification of the copolymer, and the molecular weight of the PSMA copolymer backbone. Butylamine and pentylamine derivatives of PSMA copolymers exhibited more than 80% hemolysis at endosomal pH values, which suggests their potential as a platform of "smart" polymeric carriers for enhanced cytoplasmic delivery of a variety of therapeutic macromolecules.

Limited cytoplasmic delivery of enzyme-susceptible drugs remains a significant challenge facing the development of protein and nucleic acid therapies that act in intracellular compartments. "Smart" pH-responsive, membrane-destabilizing polymers present a new approach to shuttling therapeutic molecules past the endosomal membrane and into the cytoplasm of targeted cells. This report describes the use of a functionalized monomer, pyridyl disulfide acrylate (PDSA), to develop pH-responsive, membrane-destabilizing, and glutathione-reactive polymers by copolymerization with several pH-responsive and hydrophobic monomers. The activity of the carriers is described as a function of (a) increasing the length of the hydrophobic alkyl group substituted onto the pH-responsive monomer and (b) the incorporation of a hydrophobic monomer such as butyl acrylate (BA) on the pH sensitivity and membrane-destabilizing activity of new polymer compositions. The membrane-destabilizing activity of different polymer compositions was evaluated as a function of pH and polymer concentration using the red blood cell (RBC) hemolysis assay. Hemolysis results show that the increase in the hydrophobic character of the polymer backbone results in a shift in the pH sensitivities and an increase in the membrane-destabilizing activity. Results show that the observed hemolytic activities and pH sensitivity profiles could be designed across a range that matches the properties needed for enhancing the cytoplasmic delivery of macromolecular therapeutic.

Many molecular imaging techniques rely on tracer methods to visualize specific physiological processes in cells, animals, and humans. A new family of "smart" delivery systems for biomolecules has opened new opportunities for the molecular imaging field. One class of polymeric carriers reversibly become membrane destabilizing in response to sharp pH changes and were designed for delivering proteins and nucleic acids to intracellular compartments. These carriers could enable the use of imaging agents and intracellular reporters whose site of action made them previously inaccessible. A second class of stimuli-responsive polymer-biomolecule conjugates can be reversibly formed into particles of closely defined sizes. The ability to control when and where the protein or DNA species is in the free versus particle form may allow imaging applications that exploit their differential size and diffusion properties.

The objective of this research was to investigate the effect of surface charge of poly(amidoamine), PAMAM, dendrimers on the integrity, paracellular permeability, and viability of Caco-2 cell monolayers by monitoring the transepithelial electrical resistance (TEER), mannitol permeability, and leakage of lactate dehydrogenase (LDH) enzyme, respectively. Neutral PAMAMOH, generations 2-4 (G2-G4), and anionic PAMAM-COOH (G-0.5-G4.5) dendrimers were incubated with Caco-2 cell monolayers at donor concentrations of 0.1, 1.0, and 10.0 mM for 90, 150, and 210 min. Neutral G2-G4 and anionic G-0.5, G0.5, G1.5 and G4.5 dendrimers did not cause any significant change in TEER or mannitol permeability across Caco-2 cell monolayers. Anionic G2.5 and G3.5 dendrimers, however, caused an incubation time-dependant decline in TEER values and up to a 6-fold increase in mannitol permeability. All anionic PAMAM-COOH dendrimers caused an incubation time-, concentration-, and generation-dependant LDH leakage that was not observed with neutral PAMAM-OH dendrimers. These studies suggest a size and/or charge “window” where anionic dendrimers may enhance paracellular transport across Caco-2 cell monolayers further confirming their potential as drug carriers and permeation enhancers for oral drug delivery.

Purpose. To study the influence of a controlled incremental increase in size and molecular weight of a series of poly(amidoamine) (PAMAM) dendrimers on their extravasation across the microvascular network endothelium.

Effective treatment of solid tumors requires homogenous distribution of anticancer drugs within the entire tumor volume to deliver lethal concentrations to resistant cancer cells and tumor-initiating cancer stem cells. However, penetration of small molecular weight chemotherapeutic agents and drug-loaded polymeric and lipid particles into the hypoxic and necrotic regions of solid tumors remains a significant challenge. This article reports the results of pulsed ultrasound enhanced penetration of nano-sized fluorescent particles into MCF-7 breast cancer spheroids (300-350 μm diameter) as a function of particle size and charge. With pulsed ultrasound application in the presence of microbubbles, small (20 nm) particles achieve 6-20 folds higher penetration and concentration in the spheroid's core compared to those not exposed to ultrasound. Increase in particle size to 40 nm and 100 nm results in their effective penetration into the spheroid's core to 9 and 3 folds, respectively. In addition, anionic carboxylate particles achieved higher penetration (2.3, 3.7, and 4.7 folds) into the core (0.25r) of MCF-7 breast cancer spheroids compared to neutral (2.2, 1.9, and 2.4 folds) and cationic particles (1.5, 1.4 and 1.9 folds) upon US exposure for 30, 60, and 90 seconds under the same experimental conditions. These results demonstrate the feasibility of utilizing pulsed ultrasound to increase the penetration of nano-sized particles into MCF-7 spheroids mimicking tumor tissue. The effects of particle properties on the penetration enhancement were also illustrated.

This report describes the design and synthesis of a new series of degradable, pH-sensitive, membrane-destabilizing, comb-like polymers that can enhance the intracellular delivery of therapeutic nucleic acids. These comb-like polymers are based on a diblock polymer backbone where the first block is a copolymer of pH-sensitive ethyl acrylic acid (EAA) monomers and hydrophobic butyl methacrylate (BMA) or hexyl methacrylate monomers. The second block is a homopolymer of N-acryloxy succinimide (NASI) or s-benzyl l -aspartate N-carboxy-anhydride (BLA-NCA) monomers, which are functionalized to allow controlled grafting of hydrophobic HMA and cationic trimethyl aminoethyl methacrylate (TMAEMA) copolymers via acid-labile hydrazone linkages. These comb-like polymers displayed high hemolytic activity in acidic solutions, which increased with the increase in polymer concentration. All comb-like polymers degraded into small fragments upon incubation in an acidic solution (pH 5.8) due to hydrolysis of the hydrazone linkages connecting the hydrophobic/cationic grafts to the polymer backbone. Comb-like polymers successfully complexed anti-GAPDH siRNA molecules into serum- and nuclease-stable particles, which successfully silenced GAPDH expression at both the mRNA and protein levels. These results collectively indicate the potential of these new comb-like polymers to serve as vehicles for effective intracellular delivery of therapeutic nucleic acids.

An excessive inflammatory response is a clinical problem following major infections and severe injury that may lead to Sepsis Syndrome and Multiple Organ Failure (MOF), including the Acute Respiratory Distress Syndrome (ARDS). Management of excessive inflammation may be possible through control of key inflammatory pathways such as those mediated by the important interleukin-1 receptor associated kinase-1 (IRAK-1). In the current study, we report the impact on gene expression induced by lipopolysaccharide (LPS) stimulation of THP-1 cells treated with an antisense oligonucleotide (ASODN) against the IRAK-1 gene using cDNA microarrays and quantitative RT-PCR. The therapeutic ASODN was delivered using a pH-sensitive, membrane-interactive polymer that destabilizes the endosomal membrane to enhance access cytoplasmic delivery in targeted cells. Following LPS stimulation, the anti-inflammatory activity of ASODN against the IRAK-1 gene expression is evidenced by the lower expression of inflammatory chemokines, cytokines and acute-phase proteins compared to control cells. These results provide a larger mechanistic picture of IRAK-1 knockdown by this polymer therapeutic in macrophage-like cells.

Purpose: Sulfated polysaccharides extract of Ulva lactuca (ULSP) was in vitro assessed as probable chemopreventive and therapeutic agents for colon cancer (CC) comparing with currently used chemotherapy (5-fluorouracil “5-FU” and mitomycin-C “MMC”) using our newly developed in vitro transformed cancerous rat colon epithelial cells. Methods: The present study used an in vitro animal CC model as it resembles human multistage of chromosomal instability type of CC that is located mainly at distal colon. Hence, this model was used for evaluating ULSP by the detection of cytotoxicity against normal and cancerous colon cells and antimetastatic potency. Besides, ULSP efficacy as CC chemopreventive agent was assessed through the detection of CC progression markers; loss of heterozygosity of deleted in colorectal cancer (DCC) and p53, aldehyde dehydrogenase (ALDH) 1B1 expression, activities of ornithine decarboxylase (ODC), cyclooxygenase-2 (COX-2) and alkaline phosphatase (ALP), cellular redox state (Eh), doubling time, and mucin level. Results: The apoptosis-dependent anticancer activity of ULSP (4.9 µg/ml) was revealed at all stages of cancerous distal colon epithelial cells by the formation of apoptotic bodies and DNA fragmentation (above 43%) without affecting normal colon epithelial cells. Also, its inhibitory effect on the invasion of cancerous epithelial and Caco-2 cells (above 49%) was stronger than that of 5-FU+MMC (about 11%). Cells were treated with ULSP before exposure to CC inducers and they showed normal tumor suppressor genes (DCC and p53), normal expression of ALDH1B1, and normal activity of ODC (0.03±0.0004 U/μg) and null activity of COX-2. In addition, their normal pattern of proliferations (doubling time 3 days and Eh 252.7±2.12 mV) and differentiation was confirmed by the maintenance of ALP activity (0.46±0.01 mU/μg) and the secretion of mucin (29.2±0.2 μg/ml) compared to normal and cancerous colon epithelial cells. Conclusion: Our innovative in vitro model of colon cancer have proved that ULSP could have induced apoptosis at all stages of cancerous colon cells, inhibited cancerous cells to metastasize and blocked CC progression without effect on normal epithelial cells comparable to chemotherapy. Citation Format: Marwa Abu-Serie, Maha El Demellawy, Mohamed El-Sayed, Fatma El-Rashidy. In vitro animal cancer model for assessment of sulfated polysaccharides extract of Ulva lactuca as colon cancer therapeutic and chemopreventive agents. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B13.

Limited cytoplasmic delivery of enzyme-susceptible drugs remains a significant challenge facing the development of protein and nucleic acid therapies that act in intracellular compartments. Researchers have examined several approaches, including fusogenic proteins and protein transduction domains, to enhance the intracellular delivery of the therapeutic cargo. This review summarises efforts to develop ‘smart’ pH-sensitive and membrane-destabilising polymers that can shuttle therapeutic peptide, protein and nucleic acid molecules past the endosomal membrane into the cytoplasm of targeted cells. Several classes of ‘smart’ non-degradable polymeric carriers have been developed that have proved effective both in vitro and in vivo in enhancing the cytoplasmic delivery of a variety of therapeutic molecules.

This article summarizes our efforts to evaluate the potential of poly (amidoamine) (PAMAM) dendrimers as carriers for oral drug delivery. Specifically, the permeability of a series of cationic PAMAM–NH2 (G0–G4) dendrimers across Caco-2 cell monolayers was evaluated as a function of dendrimer generation, concentration, and incubation time. The influence of dendrimer surface charge on the integrity, paracellular permeability, and viability of Caco-2 cell monolayers was monitored by measuring the transepithelial electrical resistance (TEER), 14C-mannitol permeability, and leakage of lactate dehydrogenase (LDH) enzyme, respectively. Microvascular extravasation of PAMAM–NH2 dendrimers in relation to their size, molecular weight, and molecular geometry is also discussed. Results of these studies show that transepithelial transport and microvascular extravasation of PAMAM dendrimers are dependent on their structural features including molecular size, molecular geometry, and surface chemistry. These results suggest that by optimizing the size and surface charge of PAMAM dendrimers, it is possible to develop oral delivery systems based on these carriers for targeted drug delivery.

The objective of this research was to investigate the mechanism(s) of transport of generation 2 (G2) poly (amidoamine) dendrimers across Caco-2 cell monolayers. The contribution of an energy-dependent process such as adsorptive endocytosis was investigated by determining G2 permeability at 4 and 37 degrees C. The contribution of P-gp efflux to transport was examined by determining the apical to basolateral (AB) and basolateral to apical (BA) permeability of 14C-paclitaxel in presence of G2, and by determining AB and BA permeability of G2 in presence of paclitaxel. The permeability of G2 and 14C-mannitol was investigated in the presence of palmitoyl carnitine to determine the contribution of the paracellular pathway. Permeability of G2 at 4 degrees C was significantly (P0.05) lower than that observed at 37 degrees C. AB and BA permeability of 14C-paclitaxel did not change in the presence of G2. AB and BA permeability of G2 did not change in the presence of paclitaxel. The permeability of G2 and 14C-mannitol increased significantly (P0.05) in the presence of palmitoyl carnitine, and in addition, 14C-mannitol permeability was increased in presence of G2. The permeability of G2 across Caco-2 cell monolayers appears to involve a combination of paracellular transport and an energy-dependent process, possibly adsorptive endocytosis. G2 dendrimers do not appear to be substrates for the P-gp efflux system.

The objective of this study was to investigate the influence of physiochemical parameters (such as size, molecular weight, molecular geometry, and number of surface amine groups) of poly (amidoamine) (PAMAM) dendrimers, on their permeability across Caco-2 cell monolayers. The permeability of a series of PAMAM dendrimers, generations 0-4 (G0-G4), was investigated across Caco-2 cell monolayers in both the apical to basolateral (AB) and basolateral to apical (BA) directions. The influence of PAMAM dendrimers on the integrity, paracellular permeability, and viability of Caco-2 cell monolayers was also monitored by measuring the transepithelial electrical resistance (TEER), mannitol permeability, and leakage of lactate dehydrogenase (LDH) enzyme, respectively. G0, G1 and G2 demonstrated similar AB permeabilities, which were moderate several fold higher than the AB permeability of higher generations. The AB and BA permeability of G0-G4 typically increased with the increase in donor concentration and incubation time. Permeability values are not reported at generations, concentrations or incubation times that the dendrimers were toxic to Caco-2 cells. TEER values decreased and mannitol permeability increased as a function of donor concentration, incubation time, and generation number. LDH results for G3 and G4 indicate that Caco-2 cell viability was reduced with increasing donor concentration, incubation time, and generation number. The appreciable permeability of G0-G2, coupled with their nontoxic effects on Caco-2 cells, suggest their potential as water-soluble polymeric drug carriers for controlled oral drug delivery.

The objective of this study was to determine the permeability of a series of poly amidoamine (PAMAM) dendrimers of generations 0-4 (G0-G4) across MDCK (Madin-Darby Canine Kidney) cell line. PAMAM dendrimers with incremental increase in size and molecular weight were labeled by fluorescein isothiocyanate (FITC) and the least polydisperse fractions were collected by size exclusion chromatography. MDCK cells were grown on Transwell filters for four days. The conjugates were detected by HPLC equipped with fluorescence detector. The permeability of the dendrimers across MDCK cells was determined in the apical to basolateral direction. The rank-order permeability of the PAMAM dendrimers was G4 >> G1 approximately G0 > G3 > G2. The permeability of mannitol in the presence of G4 increased by nine-fold. Results suggest that the transepithelial transport of PAMAM dendrimers is effected by both the polymer size, and the modulation of the cell membrane by the cationic dendrimers.

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.

Poly(amidoamine) (PAMAM) dendrimers are a family of water-soluble polymers with a characteristic tree-like branching architecture and a large number of surface groups, which have been used to immobilize a variety of therapeutic molecules for targeted drug delivery. Earlier studies showed that small cationic PAMAM-NH2 and selected anionic PAMAM-COOH dendrimers permeate across in vitro models of the small intestinal epithelium by paracellular and transcellular transport mechanisms. The focus of this research is to mathematically calculate the effect of cationic, anionic, and neutral PAMAM dendrimers on the porosity of epithelial tight junctions as a function of dendrimers concentration, incubation time, generation number, and charge density. Results show that the increase in the concentration, incubation time and generation number of cationic G0-G2 PAMAM-NH2 and anionic G2.5 and G3.5 PAMAM-COOH dendrimers caused a corresponding increase in the porosity of Caco-2 cell monolayers. Neutral G2-G4 PAMAM-OH dendrimers had no effect on the porosity of intestinal cells. These results provide quantitative evidence that the observed increase in permeability of PAMAM dendrimers across Caco-2 cell monolayers is due to their effect on the organization of the tight junctions and the associated increase in membrane porosity. Furthermore, these results emphasize the potential of cationic PAMAM-NH2 and anionic PAMAM-COOH dendrimers to function as carriers for controlled oral drug delivery.