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1.  Stability Analysis of Glutamic Acid Linked Peptides Coupled to NOTA through Different Chemical Linkages 
Molecular Pharmaceutics  2014;11(11):3867-3874.
Glutamic acid is a commonly used linker to form dimeric peptides with enhanced binding affinity than their corresponding monomeric counterparts. We have previously labeled NOTA-Bn-NCS-PEG3-E[c(RGDyK)]2 (NOTA-PRGD2) [1] with [18F]AlF and 68Ga for imaging tumor angiogenesis. The p-SCN-Bn-NOTA was attached to E[c(RGDyK)]2 [2] through a mini-PEG with a thiourea linkage, and the product [1] was stable at radiolabeling condition of 100 °C and pH 4.0 acetate buffer. However, when the same p-SCN-Bn-NOTA was directly attached to the α-amine of E[c(RGDfK)]2 [3], the product NOTA-Bn-NCS-E[c(RGDfK)]2 [4] became unstable under similar conditions and the release of monomeric c(RGDfK) [5] was observed. The purpose of this work was to use HPLC and LC-MS to monitor the decomposition of glutamic acid linked dimeric peptides and their NOTA derivatives. A c(RGDyK) [6] and bombesin (BBN) [7] heterodimer c(RGDyK)-E-BBN [8], and a dimeric bombesin E(BBN)2 [9], both with a glutamic acid as the linker, along with a model compound PhSCN-E[c(RGDfK)] [10] were also studied. All the compounds were dissolved in 0.5 M pH 4.0 acetate buffer at the concentration of 1 mg/mL, and 0.1 mL of each sample was heated at 100 °C for 10 min and the more stable compounds were heated for another 30 min. The samples at both time points were analyzed with analytical HPLC to monitor the decomposition of the heated samples. The samples with decomposition were further analyzed by LC-MS to determine the mass of products from the decomposition for possible structure elucidation. After 10 min heating, the obvious release of c(RGDfK) [5] was observed for NOTA-Bn-NCS-E[c(RGDfK)]2 [4] and Ph-SCN-E[c(RGDfK)] [10]. Little or no release of monomers was observed for the remaining samples at this time point. After further heating, the release of monomers was clearly observed for E[c(RGDyK)]2 [2], E[c(RGDfK)]2 [3], c(RGDyK)-E-BBN [8], and E(BBN)2 [9]. No decomposition or little decomposition was observed for NOTA-Bn-NCS-PEG3-E[c(RGDyK)]2 [1], PEG3-E[c(RGDyK)]2 [11], NOTA-E[c(RGDyK)]2 [12], and NOTA-PEG3-E[c(RGDyK)]2 [13]. The glutamic acid linked dimeric peptides with a free α-amine are labile due to the neighboring amine participation in the hydrolysis. The stability of peptides could be increased by converting the free amine into amide. The instability of thiourea derivatives formed from α-amine was caused by participation of thiol group derived from thiourea.
doi:10.1021/mp400706q
PMCID: PMC4224566  PMID: 24533430
peptide; glutamate linker; thiourea; hydrolysis; Edman degradation
2.  99mTc-Labeled Cystine Knot Peptide Targeting Integrin αvβ6 for Tumor SPECT Imaging 
Molecular Pharmaceutics  2014;11(4):1208-1217.
Integrin αvβ6 is overexpressed in a variety of cancers, and its expression is often associated with poor prognosis. Therefore, there is a need to develop affinity reagents for noninvasive imaging of integrin αvβ6 expression since it may provide early cancer diagnosis, more accurate prognosis, and better treatment planning. We recently engineered and validated highly stable cystine knot peptides that selectively bind integrin αvβ6 with no cross-reactivity to integrins αvβ5, α5β1, or αvβ3, also known to be overexpressed in many cancers. Here, we developed a single photon emission computed tomography (SPECT) probe for imaging integrin αvβ6 positive tumors. Cystine knot peptide, S02, was first conjugated with a single amino acid chelate (SAAC) and labeled with [99mTc(H2O)3(CO)3]+. The resulting probe, 99mTc-SAAC-S02, was then evaluated by in vitro cell uptake studies using two αvβ6 positive cell lines (human lung adenocarcinoma cell line HCC4006 and pancreatic cancer cell line BxPC-3) and two αvβ6 negative cell lines (human lung adenocarcinoma cell line H838 and human embryonic kidney cell line 293T). Next, SPECT/CT and biodistribution studies were performed in nude mice bearing HCC4006 and H838 tumor xenografts to evaluate the in vivo performance of 99mTc-SAAC-S02. Significant differences in the uptake of 99mTc-SAAC-S02 were observed in αvβ6 positive vs negative cells (P < 0.05). Biodistribution and small animal SPECT/CT studies revealed that 99mTc-SAAC-S02 accumulated to moderate levels in antigen positive tumors (∼2% ID/g at 1 and 6 h postinjection, n = 3 or 4/group). Moreover, the probe demonstrated tumor-to-background tissue ratios of 6.81 ± 2.32 (tumor-to-muscle) and 1.63 ± 0.18 (tumor-to-blood) at 6 h postinjection in αvβ6 positive tumor xenografts. Co-incubation of the probe with excess amount of unlabeled S02 as a blocking agent demonstrated significantly reduced tumor uptake, which is consistent with specific binding to the target. Renal filtration was the main route of clearance. In conclusion, knottin peptides are excellent scaffolds for which to develop highly stable imaging probes for a variety of oncological targets. 99mTc-SAAC-S02 demonstrates promise for use as a SPECT agent to image integrin αvβ6 expression in living systems.
doi:10.1021/mp400683q
PMCID: PMC3993876  PMID: 24524409
integrin αvβ6; cystine-knot peptide; 99mTc; SPECT
3.  Injection Route and TLR9 Agonist Addition Significantly Impact Heroin Vaccine Efficacy 
Molecular Pharmaceutics  2014;11(3):1075-1080.
Active immunization is an effective means of blocking the pharmacodynamic effects of drugs and holds promise as a treatment for heroin addiction. Previously, we demonstrated the efficacy of our first-generation vaccine in blocking heroin self-administration in rats, however, many vaccine components can be modified to further improve performance. Herein we examine the effects of varying heroin vaccine injection route and adjuvant formulation. Mice immunized via subcutaneous (sc) injection exhibited inferior anti-heroin titers compared to intraperitoneal (ip) and sc/ip coadministration injection routes. Addition of TLR9 agonist cytosine-guanine oligodeoxynucleotide 1826 (CpG ODN 1826) to the original alum adjuvant elicited superior antibody titers and opioid affinities compared to alum alone. To thoroughly assess vaccine efficacy, full dose–response curves were generated for heroin-induced analgesia in both hot plate and tail immersion tests. Mice treated with CpG ODN 1826 exhibited greatly shifted dose–response curves (10–13-fold vs unvaccinated controls) while non-CpG ODN vaccine groups did not exhibit the same robust effect (2–7-fold shift for ip and combo, 2–3-fold shift for sc). Our results suggest that CpG ODN 1826 is a highly potent adjuvant, and injection routes should be considered for development of small molecule–protein conjugate vaccines. Lastly, this study has established a new standard for assessing drugs of abuse vaccines, wherein a full dose–response curve should be performed in an appropriate behavioral task.
doi:10.1021/mp400631w
PMCID: PMC3993894  PMID: 24517171
vaccine; heroin; analgesia; dose−response; antibody; route of administration; adjuvant; CpG ODN
4.  [No title available] 
PMCID: PMC3993937  PMID: 24495091
5.  [No title available] 
PMCID: PMC4096228  PMID: 24495169
6.  [No title available] 
PMCID: PMC3993913  PMID: 24484077
7.  [No title available] 
PMCID: PMC4016717  PMID: 24294970
8.  [No title available] 
PMCID: PMC4031264  PMID: 24320221
9.  [No title available] 
PMCID: PMC4031276  PMID: 24328955
10.  [No title available] 
PMCID: PMC4031282  PMID: 24377350
11.  [No title available] 
PMCID: PMC4031293  PMID: 24364805
12.  Evaluation of glycodendron and synthetically-modified dextran clearing agents for multi-step targeting of radioisotopes for molecular imaging and radioimmunotherapy 
Molecular pharmaceutics  2013;11(2):400-416.
A series of N-acetylgalactosamine-dendrons (NAG-dendrons) and dextrans bearing biotin moieties were compared for their ability to complex with and sequester circulating bispecific anti-tumor antibody (scFv4) streptavidin (SA) fusion protein (scFv4-SA) in vivo, to improve tumor to normal tissue concentration ratios for targeted radioimmunotherapy and diagnosis. Specifically, a total of five NAG-dendrons employing a common synthetic scaffold structure containing 4, 8, 16, or 32 carbohydrate residues and a single biotin moiety were prepared (NAGB), and for comparative purposes, a biotinylated-dextran with average molecular weight (MW) of 500 kD was synthesized from amino-dextran (DEXB). One of the NAGB compounds, CA16, has been investigated in humans; our aim was to determine if other NAGB analogs (e.g. CA8 or CA4) were bioequivalent to CA16 and/or better suited as MST reagents. In vivo studies included dynamic positron-emission tomography (PET) imaging of 124I-labelled-scFv4-SA clearance and dual-label biodistribution studies following multi-step targeting (MST) directed at subcutaneous (s.c.) human colon adenocarcinoma xenografts in mice. The MST protocol consists of three injections: first, a bispecific antibody specific for an anti-tumor associated glycoprotein (TAG-72) single chain genetically-fused with SA (scFv4-SA); second, CA16 or other clearing agent; and third, radiolabeled biotin. We observed using PET imaging of 124I-labelled-scFv4-SA clearance that the spatial arrangement of ligands conjugated to NAG (i.e. biotin) can impact the binding to antibody in circulation and subsequent liver uptake of the NAG-antibody complex. Also, NAGB CA32-LC or CA16-LC can be utilized during MST to achieve comparable tumor- to-blood ratios and absolute tumor uptake seen previously with CA16. Finally, DEXB was equally effective as NAGB CA32-LC at lowering scFv4-SA in circulation, but at the expense of reducing absolute tumor uptake of radiolabeled biotin.
doi:10.1021/mp4003128
PMCID: PMC4270281  PMID: 24219178
asialoglycoprotein; pretargeting; radioimmunotherapy; bispecific antibodies
13.  Toward a Rationale for the PTC124 (Ataluren) Promoted Readthrough of Premature Stop Codons: A Computational Approach and GFP-Reporter Cell-Based Assay 
Molecular Pharmaceutics  2014;11(3):653-664.
The presence in the mRNA of premature stop codons (PTCs) results in protein truncation responsible for several inherited (genetic) diseases. A well-known example of these diseases is cystic fibrosis (CF), where approximately 10% (worldwide) of patients have nonsense mutations in the CF transmembrane regulator (CFTR) gene. PTC124 (3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)-benzoic acid), also known as Ataluren, is a small molecule that has been suggested to allow PTC readthrough even though its target has yet to be identified. In the lack of a general consensus about its mechanism of action, we experimentally tested the ability of PTC124 to promote the readthrough of premature termination codons by using a new reporter. The reporter vector was based on a plasmid harboring the H2B histone coding sequence fused in frame with the green fluorescent protein (GFP) cDNA, and a TGA stop codon was introduced in the H2B-GFP gene by site-directed mutagenesis. Additionally, an unprecedented computational study on the putative supramolecular interaction between PTC124 and an 11-codon (33-nucleotides) sequence corresponding to a CFTR mRNA fragment containing a central UGA nonsense mutation showed a specific interaction between PTC124 and the UGA codon. Altogether, the H2B-GFP-opal based assay and the molecular dynamics (MD) simulation support the hypothesis that PTC124 is able to promote the specific readthrough of internal TGA premature stop codons.
doi:10.1021/mp400230s
PMCID: PMC4167060  PMID: 24483936
cystic fibrosis (CF); Duchenne muscular distrophy (DMD); premature termination codons (PTC); nonsense mutation readthrough; oxadiazoles; ataluren; molecular dynamics (MD); green fluorescent protein (GFP)
14.  Inhibiting Metastatic Breast Cancer Cell Migration via the Synergy of Targeted, pH-triggered siRNA Delivery and Chemokine Axis Blockade 
Molecular Pharmaceutics  2014;11(3):755-765.
Because breast cancer patient survival inversely correlates with metastasis, we engineered vehicles to inhibit both the C-X-C chemokine receptor type 4 (CXCR4) and lipocalin-2 (Lcn2) mediated migratory pathways. pH-responsive liposomes were designed to protect and trigger the release of Lcn2 siRNA. Liposomes were modified with anti-CXCR4 antibodies to target metastatic breast cancer (MBC) cells and block migration along the CXCR4-CXCL12 axis. This synergistic approach—coupling the CXCR4 axis blockade with Lcn2 silencing—significantly reduced migration in triple-negative human breast cancer cells (88% for MDA-MB-436 and 92% for MDA-MB-231). The results suggested that drug delivery vehicles engineered to attack multiple migratory pathways may effectively slow progression of MBC.
doi:10.1021/mp4004699
PMCID: PMC3993942  PMID: 24467226
CXCR4; liposome; siRNA; breast cancer; lipocalin-2; migration
15.  The Absence of CpG in Plasmid DNA–Chitosan Polyplexes Enhances Transfection Efficiencies and Reduces Inflammatory Responses in Murine Lungs 
Molecular Pharmaceutics  2014;11(3):1022-1031.
Chitosan polyplexes containing plasmid DNA (pDNA) have significant potential for pulmonary gene delivery applications. However, prior to using chitosan/pDNA polyplexes (CSpp) in clinical applications, their potential cytotoxicity needs to be investigated. In this study, we formulated 200–400 nm CSpp with amine to phosphate (N/P) ratios that ranged from 1 to 100. We compared two types of plasmids within CSpp: pDNA that was free of CpG sequences (CpG(−)) and pDNA that contained CpG sequences (CpG(+)). Both forms of CSpp showed low cytotoxicity when cultured with A549 and HEK293 cell lines in vitro. CSpp(CpG(−)) generated higher luciferase expression both in vitro, for A549 cells, and in vivo, compared with CSpp(CpG(+)). In addition, CSpp(CpG(−)) elicited milder inflammatory responses in mice one day subsequent to nasal instillation, as determined by proinflammatory cytokine levels within the bronchoalveolar lavage fluid. Our findings suggest that to achieve optimal gene expression with minimal cytotoxicity, inflammation, and oxidative stress, the N/P ratios and CpG sequences in the pDNA of CSpp need to be considered. These findings will inform the preclinical safety assessments of CSpp in pulmonary gene delivery systems.
doi:10.1021/mp400689r
PMCID: PMC3993893  PMID: 24494979
chitosan; plasmid DNA; polyplexes; nanoparticles; gene delivery; lung inflammation; transfection; luciferase; CpG
16.  Enhanced Cellular Entry and Efficacy of Tat Conjugates by Rational Design of the Auxiliary Segment 
Molecular Pharmaceutics  2014;11(3):964-973.
Conjugation with a cell penetrating peptide such as Tat presents an effective approach to improve the intracellular accumulation of molecules with low membrane permeability. This strategy, however, leads to a reduced cellular entry of molecules that can cross cell membrane effectively. We report here that covalent linkage of an additional hydrophobic unit that mimics a hydrophobic domain near the Tat sequence can further improve the cellular uptake of the parental conjugate into cancer cells regardless of the membrane permeability of the unconjugated molecule. Both fluorescent imaging and flow cytometry measurements confirmed the effect of palmitoylation on the increased internalization of the Tat conjugates with either 5-carboxyfluorescein (5-FAM), a nonmembrane penetrating dye, or doxorubicin, an anticancer cancer drug that can readily diffuse across cell membranes. In the case of the Tat–doxorubicin conjugate, palmitoylation improves the conjugate’s anticancer activity in both drug sensitive and resistant cervical cancer cell lines. We further demonstrate that modification of a Tat–5-FAM conjugate with a hydrophobic quencher could not only efficiently quench the fluorescence outside of cancer cell but also facilitate its entry into MCF-7 breast cancer cells. These results highlight the importance of rational molecular design of using peptide conjugation chemistry in cancer therapeutics and diagnostics.
doi:10.1021/mp400619v
PMCID: PMC3993903  PMID: 24437690
cell penetrating peptide; nanobeacon; multidrug resistant; cellular uptake
17.  Treatment of Experimental Autoimmune Encephalomyelitis by Codelivery of Disease Associated Peptide and Dexamethasone in Acetalated Dextran Microparticles 
Molecular Pharmaceutics  2014;11(3):828-835.
Multiple sclerosis (MS) is an autoimmune, demyelinating disease of the central nervous system that can cause loss of motor function and is thought to result, in part, from chronic inflammation due to an antigen-specific T cell immune response. Current treatments suppress the immune system without antigen specificity, increasing the risks of cancer, chronic infection, and other long-term side effects. In this study, we show treatment of experimental autoimmune encephalomyelitis (EAE), a model of MS, by coencapsulating the immunodominant peptide of myelin oligodendrocyte glycoprotein (MOG) with dexamethasone (DXM) into acetalated dextran (Ac-DEX) microparticles (DXM/MOG/MPs) and administering the microparticles subcutaneously. The clinical score of the mice was reduced from 3.4 to 1.6 after 3 injections 3 days apart with the coencapsulated microparticulate formulation (MOG 17.6 μg and DXM 8 μg). This change in clinical score was significantly greater than observed with phosphate-buffered saline (PBS), empty MPs, free DXM and MOG, DXM/MPs, and MOG/MPs. Additionally, treatment with DXM/MOG/MPs significantly inhibited disease-associated cytokine (e.g., IL-17, GM-CSF) expression in splenocytes isolated in treated mice. Here we show a promising approach for the therapeutic treatment of MS using a polymer-based microparticle delivery platform.
doi:10.1021/mp4005172
PMCID: PMC3993881  PMID: 24433027
multiple sclerosis; microparticle; immunotherapy; acetalated dextran
18.  Grafting Aptamers onto Gold Nanostars Increases in Vitro Efficacy in a Wide Range of Cancer Cell Types 
Molecular Pharmaceutics  2014;11(2):580-587.
We report the design of a nanoconstruct that can function as a cell-type independent agent by targeting the ubiquitous protein nucleolin. Gold nanostars (AuNS) loaded with high densities of nucleolin-specific DNA aptamer AS1411 (Apt-AuNS) produced anticancer effects in a panel of 12 cancer lines containing four representative subcategories. We found that the nanoconstructs could be internalized by cancer cells and trafficked to perinuclear regions. Apt-AuNS resulted in downregulation of antiapoptotic Bcl-2 mRNA expression by ca. 200% compared to cells without the nanoconstructs. The caspase 3/7 activity (apoptosis) and cell death in cancer cells treated with Apt-AuNS increased by 1.5 times and by ca. 17%, respectively, compared to cells treated with free AS1411 at over 10 times the concentration. Moreover, light-triggered release of aptamer from the AuNS further enhanced the in vitro efficacy of the nanoconstructs in the cancer line panel with a 2-fold increase in caspase activity and a 40% decrease in cell viability compared to treatment with Apt-AuNS only. In contrast, treatments of the nanoconstructs with or without light-triggered release on a panel of normal cell lines had no adverse effects.
doi:10.1021/mp4005657
PMCID: PMC3974612  PMID: 24422969
aptamers; gold nanostars; nucleolin; anticancer agents
19.  A Nitric Oxide-Releasing Heparin Conjugate for Delivery of a Combined Antiplatelet/Anticoagulant Agent 
Molecular Pharmaceutics  2014;11(2):645-650.
Heparin is a widely used anticoagulant due to its ability to inhibit key components in the coagulation cascade such as Factor Xa and thrombin (Factor IIa). Its potential to preferentially bind to antithrombin (ATIII) results in a conformational change and activation that leads to the prevention of fibrin formation from fibrinogen and ultimately obstructs a hemostatic plug from forming. Nitric oxide (NO) exhibits potent antiplatelet activity attributed to its capacity to increase the amount of cyclic guanosine monophosphate (cGMP) within platelets, which decreases the Ca2+ concentration required for platelet activation. Currently there is no single agent that combines the functions of both antiplatelet and anticoagulant (anti-Xa and anti-IIa) activities to effectively block both the extrinsic and the intrinsic coagulation pathways. The research reported herein demonstrates the ability to combine the physiological capabilities of both heparin and NO into one functional compound via use of a spermine derivative of heparin, thus enabling formation of a novel diazeniumdiolate (NONOate). The heparin–spermine NONOate has a half-life of 85 min at 25 °C (pH 7.4). The heparin backbone of the conjugate maintains its anticoagulant activity as demonstrated via an anti-Xa assay, providing an anticoagulant conversion of 3.6 μg/mL of the heparin–spermine–NONO conjugate being equivalent to 2.5 μg/mL (0.50 IU/mL) of underivatized heparin in terms of anti-Xa activity. Using standard platelet aggregometry, it is shown that the functionality of the NO release portion of the heparin conjugate prevents (nearly 100%) platelet aggregation in the presence of adenosine diphosphate (ADP, platelet agonist).
doi:10.1021/mp400501c
PMCID: PMC3993940  PMID: 24423090
nitric oxide release; diazeniumdiolated heparin; combined antiplatelet/anticoagulant agent
20.  An Experimental Approach To Evaluate the Impact of Impaired Transport Function on Hepatobiliary Drug Disposition Using Mrp2-Deficient TR– Rat Sandwich-Cultured Hepatocytes in Combination with Bcrp Knockdown 
Molecular Pharmaceutics  2014;11(3):766-775.
Breast cancer resistance protein (BCRP) and multidrug resistance-associated protein 2 (MRP2) are members of the ATP binding cassette (ABC) transporter family located in the canalicular membrane of hepatocytes that mediate biliary excretion of many drugs and endogenous compounds. BCRP and MRP2 have overlapping substrate profiles. Predicting drug disposition in the setting of altered transport function has important clinical significance. This investigation was designed to establish an in vitro model system to evaluate the impact of impaired Mrp2 and Bcrp function on hepatobiliary drug disposition. To achieve Bcrp knockdown by RNA interference (RNAi), sandwich-cultured hepatocytes (SCH) from Mrp2-deficient (TR–) and wild-type (WT) rats were infected with adenoviral vectors to express shRNA targeting Bcrp (Ad-siBcrp) at multiplicity of infection (MOI) of 1–10. MOI of 5 was identified as optimal. At MOI of 5, viral infection as well as WT or TR– status was statistically significant predictors of the rosuvastatin (RSV) biliary excretion index (BEI), consistent with the known role of Bcrp and Mrp2 in the biliary excretion of RSV in vivo in rats. Relative to WT rat SCH, marginal mean BEI (%) of RSV in TR– rat SCH decreased by 28.6 (95% CI: 5.8–51.3). Ad-siBcrp decreased marginal mean BEI (%) of RSV by 13.3 (7.5–9.1) relative to SCH infected with adenoviral vectors expressing a nontargeting shRNA (Ad-siNT). The BEI of RSV was almost ablated in TR– rat SCH with Bcrp knockdown (5.9 ± 3.0%) compared to Ad-siNT-infected WT rat SCH (45.4 ± 6.6%). These results demonstrated the feasibility of Bcrp knockdown in TR– rat SCH as an in vitro system to assess the impact of impaired Bcrp and Mrp2 function. At MOI of 5, viral infection had minimal effects on RSV total accumulation, but significantly decreased marginal mean taurocholate total accumulation (pmol/mg of protein) and BEI (%) by 9.9 (7.0–12.8) and 7.5 (3.7–11.3), respectively, relative to noninfected SCH. These findings may be due to off-target effects on hepatic bile acid transporters, even though no changes in protein expression levels of the hepatic bile acid transporters were observed. This study established a strategy for optimization of the knockdown system, and demonstrated the potential use of RNAi in SCH as an in vitro tool to predict altered hepatobiliary drug disposition when canalicular transporters are impaired.
doi:10.1021/mp400471e
PMCID: PMC3993909  PMID: 24410402
breast cancer resistance protein (BCRP/Bcrp); multidrug resistance-associated protein 2 (MRP2/Mrp2); RNA interference (RNAi); sandwich-cultured hepatocytes (SCH)
21.  Ultrasound-Mediated Destruction of LHRHa Targeted and Paclitaxel Loaded Lipid Microbubbles Induces Proliferation Inhibition and Apoptosis in Ovarian Cancer Cells 
Molecular pharmaceutics  2013;11(1):40-48.
Although paclitaxel (PTX) is used with platinum as the first line chemotherapy regimen for ovarian cancer, its clinical efficacy is often limited by severe adverse effects. Ultrasound targeted microbubble destruction (UTMD) technique holds a great promise in minimizing the side effects and maximizing the therapeutic efficacy. However, the technique typically uses non-targeted microbubbles with suboptimal efficiency. We synthesized targeted and PTX-loaded microbubbles (MBs) for UTMD mediated chemotherapy in ovarian cancer cells. PTX-loaded lipid MBs were coated with a luteinizing hormone-releasing hormone analogue (LHRHa) through a biotin-avidin linkage to target the ovarian cancer A2780/DDP cells that express the LHRH receptor. In the cell culture studies, PTX-loaded and LHRHa targeted MBs (TPLMBs) in combination with ultrasound (300 kHz, 0.5 W/cm2, 30 seconds) demonstrated anti-proliferative activities of 41.30 ± 3.93%, 67.76 ± 2.45%, and 75.93 ± 2.81% at 24 hours, 48 hours, and 72 hours after the treatment, respectively. The cell apoptosis ratio at 24 hours after the treatment is 32.6 ± 0.79 %, which is significantly higher than other treatment groups such as PTX only and no-targeted PTX-loaded MBs (NPLMBs) with or without ultrasound mediation. Our experiment verifies the hypothesis that ultrasound mediation of ovarian cancer targeted and drug loaded MBs will enhance the PTX therapeutic efficiency.
doi:10.1021/mp4005244
PMCID: PMC3903397  PMID: 24266423
Paclitaxel; ultrasound; microbubble; ovarian cancer; apoptosis
22.  Delivery of Chemically Glycosylated Cytochrome c Immobilized in Mesoporous Silica Nanoparticles Induces Apoptosis in HeLa Cancer Cells 
Molecular pharmaceutics  2013;11(1):102-111.
Cytochrome c (Cyt c) is a small mitochondrial heme protein involved in the intrinsic apoptotic pathway. Once Cyt c is released into the cytosol, the caspase mediated apoptosis cascade is activated resulting in programmed cell death. Herein, we explore the covalent immobilization of Cyt c into mesoporous silica nanoparticles (MSN) to generate a smart delivery system for intracellular drug delivery to cancer cells aiming at affording subsequent cell death. Cyt c was modified with sulfosuccinimidyl-6-[3′-(2-pyridyldithio)-propionamido] hexanoate (SPDP) and incorporated into SH-functionalized MSN by thiol-disulfide interchange. Unfortunately, delivery of Cyt c from the MSN was not efficient in inducing apoptosis in human cervical cancer HeLa cells. We tested whether chemical Cyt c glycosylation could be useful in overcoming the efficacy problems by potentially improving Cyt c thermodynamic stability and reducing proteolytic degradation. Cyt c lysine residues were modified with lactose at a lactose-to-protein molar ratio of 3.7±0.9 using mono-(lactosylamido)-mono-(succinimidyl) suberate linker chemistry. Circular dichroism (CD) spectra demonstrated that part of the activity loss of Cyt c was due to conformational changes upon its modification with the SPDP linker. These conformational changes were prevented in the glycoconjugate. In agreement with the unfolding of Cyt c by the linker, a proteolytic assay demonstrated that the Cyt c-SPDP conjugate was more susceptible to proteolysis than Cyt c. Attachment of the four lactose molecules reversed this increased susceptibility and protected Cyt c from proteolytic degradation. Furthermore, a cell-free caspase-3 assay revealed 47% and 87% of relative caspase activation by Cyt c-SPDP and the Cyt c-lactose bioconjugate, respectively, when compared to Cyt c. This again demonstrates the efficiency of the glycosylation to improve maintaining Cyt c structure and thus function. To test for cytotoxicity, HeLa cells were incubated with Cyt c loaded MSN at different Cyt c concentrations (12.5, 25.0, and 37.5 μg/mL) for 24 to 72 h and cellular metabolic activity determined by a cell proliferation assay. While MSN-SPDP-Cyt c did not induced cell death, the Cyt c-lactose bioconjugate induced significant cell death after 72 h, reducing HeLa cell viability to 67% and 45% at the 25 μg/mL and 37.5 μg/mL concentrations, respectively. Confocal microscopy confirmed that the MSN immobilized Cyt c-lactose bioconjugate was internalized by HeLa cells and that the bioconjugate was capable of endosomal escape. The results clearly demonstrate that chemical glycosylation stabilized Cyt c upon formulation of a smart drug delivery system and upon delivery into cancer cells and highlight the general potential of chemical protein glycosylation to improve the stability of protein drugs.
doi:10.1021/mp400400j
PMCID: PMC3905321  PMID: 24294910
Apoptosis; chemical glycosylation; drug delivery; nanoparticle; protein drug; protein stability; smart release
23.  Ultrasound-Mediated Destruction of LHRHa Targeted and Paclitaxel Loaded Lipid Microbubbles for the Treatment of Intraperitoneal Ovarian Cancer Xenografts 
Molecular pharmaceutics  2013;11(1):49-58.
Ultrasound-targeted microbubble destruction (UTMD) is a promising technique to facilitate the delivery of chemotherapy in cancer treatment. However, the process typically uses non-specific microbubbles, leading to low tumor-to-normal tissue uptake ratio and adverse side effects. In this study, we synthesized the LHRH receptor targeted and paclitaxel (PTX) loaded lipid microbubbles (TPLMBs) for tumor-specific binding and enhanced therapeutic effect at the tumor site. An ovarian cancer xenograft model was established by injecting A2780/DDP cells intraperitoneally in BALB/c nude mice. Microscopic imaging of tumor sections after intraperitoneal injection of TPLMBs showed effective binding of the microbubbles with cancer cells. Ultrasound mediated destruction of the intraperitoneally injected TPLMBs yielded a superior therapeutic outcome in comparison with other treatment options. Immunohistochemical analyses of the dissected tumor tissue further confirmed the increased tumor apoptosis and reduced angiogenesis. Our experiment suggests that ultrasound mediated intraperitoneal administration of the targeted drug-loaded microbubbles may be a useful method for the treatment of ovarian cancer.
doi:10.1021/mp400523h
PMCID: PMC3899929  PMID: 24237050
Microbubbles; ovarian cancer; intraperitoneal injection; ultrasound-targeted microbubble destruction; paclitaxel; luteinizing hormone-releasing hormone
24.  Insights into Atomic-level Interaction between Mefenamic Acid and Eudragit® EPO in a Supersaturated Solution by High-Resolution Magic-Angle Spinning NMR Spectroscopy 
Molecular pharmaceutics  2013;11(1):351-357.
The intermolecular interaction between mefenamic acid (MFA), a poorly water-soluble non-steroidal anti-inflammatory drug, and Eudragit® EPO (EPO), a water-soluble polymer, is investigated in their supersaturated solution using high-resolution magic-angle spinning (HRMAS) nuclear magnetic resonance (NMR) spectroscopy. The stable supersaturated solution with a high MFA concentration of 3.0 mg/mL is prepared by dispersing the amorphous solid dispersion into a d-acetate buffer at pH 5.5 and 37 °C. By virtue of MAS at 2.7 kHz, the extremely broad and unresolved 1H resonances of MFA in one-dimensional 1H NMR spectrum of the supersaturated solution are well resolved, thus enabling the complete assignment of MFA 1H resonances in the aqueous solution. Two-dimensional (2D) 1H/1H nuclear Overhauser effect spectroscopy (NOESY) and radio frequency-driven recoupling (RFDR) under MAS conditions reveal the interaction of MFA with EPO in the supersaturated solution at an atomic level. The strong cross-correlations observed in the 2D 1H/1H NMR spectra indicate a hydrophobic interaction between the aromatic group of MFA and the backbone of EPO. Furthermore, the aminoalkyl group in the side chain of EPO forms a hydrophilic interaction, which can be either electrostatic or hydrogen bonding, with the carboxyl group of MFA. We believe these hydrophobic and hydrophilic interactions between MFA and EPO molecules play a key role in the formation of this extremely stable supersaturated solution. In addition, 2D 1H/1H RFDR demonstrates that the molecular MFA-EPO interaction is quite flexible and dynamic.
doi:10.1021/mp4005723
PMCID: PMC3903815  PMID: 24283196
Eudragit® EPO; supersaturated solution; 1H NMR; NOESY; RFDR; intermolecular interaction
25.  Hyaluronic acid graft polymers displaying peptide antigen modulate dendritic cell response in vitro 
Molecular pharmaceutics  2013;11(1):367-373.
A novel oxime grafting scheme was utilized to conjugate an ICAM-1 ligand (LABL), a cellular antigen ovalbumin (OVA), or both peptides simultaneously to hyaluronic acid (HA). Samples of HA only and the various peptide grafted HA were found to bind to dendritic cells (DCs). HA with grafted LABL and OVA showed the greatest binding to DCs. Dendritic cells treated with HA, HA with grafted LABL, or HA with grafted LABL and OVA, significantly suppressed T cell and DC conjugate formation, T cell proliferation and reduced proinflammatory cytokine production compared to untreated cells. These results suggest that HA serves as an effective backbone for multivalent ligand presentation for inhibiting T cell response to antigen presentation. In addition, multivalent display of both antigen and an ICAM-1inhibitor (LABL) may enhance binding to DCs and could potentially disrupt cellular signaling leading to autoimmunity.
doi:10.1021/mp4003909
PMCID: PMC3927369  PMID: 24283935
peptides; hyaluronic acid; targeted delivery; dendritic cells; T cells

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