RESUMEN
Nanoscale colloidal self-assembly is an exciting approach to yield superstructures with properties distinct from those of individual nanoparticles. However, the bottom-up self-assembly of 3D nanoparticle superstructures typically requires extensive chemical functionalization, harsh conditions, and a long preparation time, which are undesirable for biomedical applications. Here, we report the directional freezing of porous silica nanoparticles (PSiNPs) as a simple and versatile technique to create anisotropic 3D superstructures with hierarchical porosity afforded by microporous PSiNPs and newly generated meso- and macropores between the PSiNPs. By varying the PSiNP building block size, the interparticle pore sizes can be readily tuned. The newly created hierarchical pores greatly augment the loading of a small molecule-anticancer drug, doxorubicin (Dox), and a large macromolecule, lysozyme (Lyz). Importantly, Dox loading into both the micro- and meso/macropores of the nanoparticle assemblies not only gave a pore size-dependent drug release but also significantly extended the drug release to 25 days compared to a much shorter 7 or 11 day drug release from Dox loaded into either the micro- or meso/macropores only. Moreover, a unique temporal drug release profile, with a higher and faster release of Lyz from the larger interparticle macropores than Dox from the smaller PSiNP micropores, was observed. Finally, the formulation of the Dox-loaded superstructures within a composite hydrogel induces prolonged growth inhibition in a 3D spheroid model of pancreatic ductal adenocarcinoma. This study presents a facile modular approach for the rapid assembly of drug-loaded superstructures in fully aqueous environments and demonstrates their potential as highly tailorable and sustained delivery systems for diverse therapeutics.
Asunto(s)
Antineoplásicos , Nanopartículas , Dióxido de Silicio/química , Porosidad , Antineoplásicos/farmacología , Nanopartículas/química , Sistemas de Liberación de Medicamentos/métodos , Doxorrubicina/farmacología , Doxorrubicina/químicaRESUMEN
Local, sustained drug delivery of potent therapeutics holds promise for the treatment of a myriad of localized diseases while eliminating systemic side effects. However, introduction of drug delivery depots such as viscous hydrogels or polymer-based implants is highly limited in stiff tissues such as desmoplastic tumors. Here, we present a method to create materials-free intratumoral drug depots through Tissue-Reactive Anchoring Pharmaceuticals (TRAPs). TRAPs diffuse into tissue and attach locally for sustained drug release. In TRAPs, potent drugs are modified with ECM-reactive groups and then locally injected to quickly react with accessible amines within the ECM, creating local drug depots. We demonstrate that locally injected TRAPs create dispersed, stable intratumoral depots deep within mouse and human pancreatic tumor tissues. TRAPs depots based on ECM-reactive paclitaxel (TRAP paclitaxel) had better solubility than free paclitaxel and enabled sustained in vitro and in vivo drug release. TRAP paclitaxel induced higher tumoral apoptosis and sustained better antitumor efficacy than the free drug. By providing continuous drug access to tumor cells, this material-free approach to sustained drug delivery of potent therapeutics has the potential in a wide variety of diseases where current injectable depots fall short.
Asunto(s)
Sistemas de Liberación de Medicamentos , Neoplasias Pancreáticas , Animales , Línea Celular Tumoral , Liberación de Fármacos , Hidrogeles , Ratones , Paclitaxel , Neoplasias Pancreáticas/tratamiento farmacológicoRESUMEN
Stimuli-responsive, on-demand release of drugs from drug-eluting depots could transform the treatment of many local diseases, providing intricate control over local dosing. However, conventional on-demand drug release approaches rely on locally implanted drug depots, which become spent over time and cannot be refilled or reused without invasive procedures. New strategies to noninvasively refill drug-eluting depots followed by on-demand release could transform clinical therapy. Here we report an on-demand drug delivery paradigm that combines bioorthogonal click chemistry to locally enrich protodrugs at a prelabeled site and light-triggered drug release at the target tissue. This approach begins with introduction of the targetable depot through local injection of chemically reactive azide groups that anchor to the extracellular matrix. The anchored azide groups then capture blood-circulating protodrugs through bioorthogonal click chemistry. After local capture and retention, active drugs can be released through external light irradiation. In this report, a photoresponsive protodrug was constructed consisting of the chemotherapeutic doxorubicin (Dox), conjugated to dibenzocyclooctyne (DBCO) through a photocleavable ortho-nitrobenzyl linker. The protodrug exhibited excellent on-demand light-triggered Dox release properties and light-mediated in vitro cytotoxicity in U87 glioblastoma cell lines. Furthermore, in a live animal setting, azide depots formed in mice through intradermal injection of activated azide-NHS esters. After i.v. administration, the protodrug was captured by the azide depots with intricate local specificity, which could be increased with multiple refills. Finally, doxorubicin could be released from the depot upon light irradiation. Multiple rounds of depot refilling and light-mediated release of active drug were accomplished, indicating that this system has the potential for multiple rounds of treatment. Taken together, these in vitro and in vivo proof of concept studies establish a novel method for in vivo targeting and on-demand delivery of cytotoxic drugs at target tissues.
Asunto(s)
Química Clic/métodos , Preparaciones de Acción Retardada , Sistemas de Liberación de Medicamentos/métodos , Liberación de Fármacos , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Doxorrubicina/administración & dosificación , Doxorrubicina/farmacocinética , Colorantes Fluorescentes , Humanos , RatonesRESUMEN
Bioorthogonal click reactions yielding stable and irreversible adducts are in high demand for in vivo applications, including in biomolecular labeling, diagnostic imaging, and drug delivery. Previously, we reported a novel bioorthogonal "click" reaction based on the coupling of ortho-acetyl arylboronates and thiosemicarbazide-functionalized nopoldiol. We now report that a detailed structural analysis of the arylboronate/nopoldiol adduct by X-ray crystallography and 11B NMR reveals that the bioorthogonal reactants form, unexpectedly, a tetracyclic adduct through the cyclization of the distal nitrogen into the semithiocarbazone leading to a strong B-N dative bond and two new 5-membered rings. The cyclization adduct, which protects the boronate unit against hydrolytic breakdown, sheds light on the irreversible nature of this polycondensation. The potential of this reaction to work in a live animal setting was studied through in vivo capture of fluorescently labeled molecules in vivo. Arylboronates were introduced into tissues through intradermal injection of their activated NHS esters, which react with amines in the extracellular matrix. Fluorescently labeled nopoldiol molecules were administered systemically and were efficiently captured by the arylboronic acids in a location-specific manner. Taken together, these in vivo proof-of-concept studies establish arylboronate/nopoldiol bioorthogonal chemistry as a candidate for wide array of applications in chemical biology and drug delivery.
Asunto(s)
Ácidos Borónicos/química , Semicarbacidas/química , Animales , Ácidos Borónicos/síntesis química , Química Clic/métodos , Cristalografía por Rayos X , Espectroscopía de Resonancia Magnética , Ratones , Modelos Moleculares , Semicarbacidas/síntesis químicaRESUMEN
Injectable alginate hydrogels have demonstrated utility in tissue engineering and drug delivery applications due in part to their mild gelation conditions, low host responses and chemical versatility. Recently, the potential of these gels has expanded with the introduction of refillable hydrogel depots - alginate gels chemically decorated with click chemistry groups to efficiently capture prodrug refills from the blood. Unfortunately, high degrees of click group substitution on alginate lead to poor viscoelastic properties and loss of ionic cross-linking. In this work, we introduce tetrabicyclononyne (tBCN) agents that covalently cross-link azide-modified alginate hydrogels for tissue engineering and drug delivery application in vivo. Adjusting cross-linker concentration allowed tuning the hydrogel mechanical properties for tissue-specific mechanical strength. The bioorthogonal and specific click reaction creates stable hydrogels with improved in vivo properties, including improved retention at injected sites. Azide-alginate hydrogels cross-linked with tBCN elicited minimal inflammation and maintained structural integrity over several months and efficiently captured therapeutics drug surrogates from the circulation. Taken together, azide-alginate hydrogels cross-linked with tBCN convey the benefits of alginate hydrogels for use in tissue engineering and drug delivery applications of refillable drug delivery depots. STATEMENT OF SIGNIFICANCE: Ionically cross-linked, injectable alginate biomaterials hold promise in many different clinical settings. However, adding new chemical functionality to alginate can disrupt their ionic cross-linking, limiting their utility. We have developed a "click" cross-linking strategy to improve the mechanical properties and tissue function of modified alginate biomaterials and enable them to capture small molecule drugs from the blood. We show that click cross-linked materials remain in place better than ionically cross-linked materials and efficiently capture payloads from the blood. Development of click cross-linking for refillable depots represents a crucial step toward clinical application of this promising drug delivery platform.
Asunto(s)
Alginatos , Hidrogeles , Materiales Biocompatibles , Química Clic , Reactivos de Enlaces Cruzados , Ingeniería de TejidosRESUMEN
Mitochondrion has emerged as one of the unconventional targets in next-generation cancer therapy. Hence, small molecules targeting mitochondria in cancer cells have immense potential in the next-generation anticancer therapeutics. In this report, we have synthesized a library of hydrazide-hydrazone-based small molecules and identified a novel compound that induces mitochondrial outer membrane permeabilization by inhibiting antiapoptotic B-cell CLL/lymphoma 2 (Bcl-2) family proteins followed by sequestration of proapoptotic cytochrome c. The new small molecule triggered programmed cell death (early and late apoptosis) through cell cycle arrest in the G2/M phase and caspase-9/3 cleavage in HCT-116 colon cancer cells, confirmed by an array of fluorescence confocal microscopy, cell sorting, and immunoblotting analysis. Furthermore, cell viability studies have verified that the small molecule rendered toxicity to a panel of colon cancer cells (HCT-116, DLD-1, and SW-620), keeping healthy L929 fibroblast cells unharmed. The novel small molecule has the potential to form a new understudied class of mitochondria targeting anticancer agent.
RESUMEN
RAS-RAF-MEK-ERK cascade in mitogen activated protein kinase (MAPK) signaling has been hijacked in colon cancer. However, the selective targeting of MAPK signaling components in colon cancer cells has remained a surmountable challenge. To address this, we have engineered hyaluronic acid cloaked 154 nm diameter oleic acid nanoparticles (HA-OA-NPs) comprising both an ERK inhibitor (AZD6244) and a DNA damaging drug (cisplatin). Dual drugs were slowly released from the HA-OA-NPs at an acidic pH (pH = 5.5) over 72 h. HCT-116 colon cancer cells engulfed these HA-OA-NPs by a CD44 receptor and clathrin-dependent endocytosis pathways followed by an accumulation into lysosomes over 6 h. These HA-OA-NPs inhibited the phosphorylation of extracellular signal-regulated kinases (ERK1 and ERK2) and damaged sub-cellular DNA to induce remarkable colon cancer cell (HCT-116 and DLD-1) death in contrast to a free drug cocktail at 24 h post incubation. Due to the biocompatibility and biodegradability of the nanoparticle components, the HA-OA-NPs could be brought into clinics as a platform for the synchronized inhibition of multiple targets for improved therapeutic efficacy in colon cancer patients.
RESUMEN
Colon cancer has emerged as one of the most devastating diseases in the whole world. Mitogen-activated protein kinase (MAPK)-phosphatidylinsitol-3-kinase (PI3K) signaling hub has gained lots of attention due to its deregulation in colon cancer cells. However, selective targeting of oncogenic MAPK-PI3K hub in colon cancer has remained highly challenging, hence it has mostly been unexplored. To address this, we have engineered a hyaluronic acid layered lipid-based chimeric nanoparticle (HA-CNP) consisting of AZD6244 (MAPK inhibitor), PI103 (PI3K inhibitor), and cisplatin (DNA impairing drug) ratiometrically in a single particle. Electron microscopy (field emission scanning electron microscopy and atomic force microscopy) and dynamic light scattering were utilized to characterize the size, shape, morphology, and surface charge of the HA-CNPs. Fluorescent confocal laser scanning microscopy and flow cytometry analysis confirmed that HA-CNPs were taken up by HCT-116 colon cancer cells by merging of clathrin and CD44 receptor-mediated endocytosis along with macropinocytosis to home into acidic organelles (lysosomes) within 1 h. A gel electrophoresis study evidently established that HA-CNPs simultaneously inhibited MAPK-PI3K signaling hub with DNA damage in HCT-116 cells. These HA-CNPs stalled the cell cycle into G0/G1 phase, leading to induction of apoptosis (early and late) in colon cancer cells. Finally, these HA-CNPs exerted remarkable cytotoxicity in HCT-116 colon cancer cells at 24 h compared to that of the free triple drug cocktail as well as HA-coated dual drug-loaded nanoparticles without showing any cell death in healthy L929 fibroblast cells. These HA-coated CNPs have potential to be translated into clinics as a novel platform to perturb various oncogenic signaling hubs concomitantly toward next-generation targeted colon cancer therapy.
RESUMEN
Breast cancer is the most devastating disease among females globally. Conventional chemotherapeutic regimen relies on the use of highly cytotoxic drugs as monotherapy and combination therapy leading to severe side effects to the patients as collateral damage. Moreover, combining hydrophobic and hydrophilic drugs create erratic biodistribution and suboptimal medicinal outcome. Hence, packaging multiple drugs of diverse mechanisms of action and biodistribution for safe delivery into tumor tissues with optimal dosages is indispensable for next-generation breast cancer therapy. To address these, in this report, we describe a unique cisplatin-triggered self-assembly of linear polymer into 3D-spherical sub 200 nm particles. These nanoparticles comprise a hydrophobic (paclitaxel) and hydrophilic drug (cisplatin) simultaneously in a single particle. Molecular dynamics simulation revealed hydrophilic-hydrophilic interaction and interchain H-bonding as underlying mechanisms of self-assembly. Confocal microscopy studies evidently demonstrated that these novel nanoparticles can home into lysosomes in breast cancer cells, fragment subcellular nuclei, and prevent cell division, leading to improved breast cancer cell death compared to free drug combination. Moreover, 3D-breast tumor spheroids were reduced remarkably by the treatment of these nanoparticles within 24 h. These dual-drug-loaded self-assembled polymeric nanoparticles have prospective to be translated into a clinical strategy for breast cancer patients.
RESUMEN
Mesoporous silica nano-channel (MCM-41) based molecular switching of a biologically important anticancer drug, namely, ellipticine (EPT) has been utilized to probe its efficient loading onto MCM-41, and its subsequent release to intra-cellular biomolecules, like DNA. By exploiting various spectroscopic techniques (like, steady state fluorescence, time-resolved fluorescence and circular dichroism), it has been shown that EPT can be easily translocated from MCM-41 to DNA without using any external stimulant. Blue emission of EPT in a polar aprotic solvent, i.e., dichloromethane (DCM), completely switches to green upon loading inside MCM-41 due to the conversion from a neutral to a protonated form of the drug inside nano-pores. Powder X-ray diffraction (PXRD), N2 gas adsorption and confocal fluorescence microscopy results confirm the adsorption of EPT inside the nano-pores of MCM-41. Here, the lysozyme (Lyz) protein has been utilized as a pore blocker of MCM-41 in order to prevent premature drug release. Interestingly, EPT is released to DNA even from the EPT-MCM-Lyz composite system, and results in intensification of green fluorescence. Electron microscopy results reveal the formation of a distinctive garland kind of morphology involving MCM-41 and DNA probably through non-covalent interactions, and this is believed to be responsible for the DNA assisted release of drug molecules from silica nano-pores. Confocal laser scanning microscopy (CLSM) imaging revealed that EPT-MCM is successfully internalized into the HeLa cervical cancer cells and localized into the nucleus. Cell viability assay results infer that EPT-MCM and EPT-MCM-Lyz showed much improved efficacy in HeLa cancer cells compared to free ellipticine.
Asunto(s)
Antineoplásicos/administración & dosificación , ADN , Liberación de Fármacos , Nanotubos , Dióxido de Silicio , Adsorción , Células HeLa , Humanos , Porosidad , Difracción de Rayos XRESUMEN
Fluorine incorporation into organic molecules imparts favorable physicochemical properties such as lipophilicity, solubility and metabolic stability necessary for drug action. Toward such applications using peptide nucleic acids (PNA), we herein report the chemical synthesis of fluorinated PNA monomers and biophysical studies of derived PNA oligomers containing fluorine in in the acetyl side chain (-CHF-CO-) bearing nucleobase uracil (5-F/5-CF3-U). The crystal structures of fluorinated racemic PNA monomers reveal interesting base pairing of enantiomers and packing arrangements directed by the chiral F substituent. Reverse phase HPLC show higher hydrophobicity of fluorinated PNA oligomers, dependent on the number and site of the fluorine substitution: fluorine on carbon adjacent to the carbonyl group induces higher lipophilicity than fluorine on nucleobase or in the backbone. The PNA oligomers containing fluorinated bases form hybrids with cDNA/RNA with slightly lower stability compared to that of unmodified aeg PNA, perhaps due to electronic effects. The uptake of fluorinated homooligomeric PNAs by HeLa cells was as facile as that of nonfluorinated PNA. In conjunction with our previous work on PNAs fluorinated in backbone and at N-terminus, it is evident that the fluorinated PNAs have potential to emerge as a new class of PNA analogues for applications in functional inhibition of RNA.
Asunto(s)
Ácidos Nucleicos de Péptidos/química , Uracilo/química , Animales , Cromatografía Líquida de Alta Presión , ADN Complementario/química , Citometría de Flujo , Fluoruros , Flúor/química , Halogenación , Células HeLa , Humanos , Enlace de Hidrógeno , Ratones , Células 3T3 NIH , ARN/química , Solubilidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Estereoisomerismo , Rayos UltravioletaRESUMEN
Phosphatidylinositol-3-kinase (PI3K) signaling has been hijacked in different types of cancers. Hence, PI3K inhibitors have emerged as novel targeted therapeutics in cancer treatment as mono and combination therapy along with other DNA damaging drugs. However, targeting PI3K signaling with small molecules leads to the emergence of drug resistance and severe side effects to the cancer patients. To address these, we have developed a biocompatible, biodegradable cholesterol-based chimeric nanoparticle (CNP), which can simultaneously load PI103, doxorubicin, and cisplatin in a controlled ratiometric manner. Size, shape, and morphology of these CNPs were characterized by dynamic light scattering (DLS), field-emission scanning electron microscopy (FESEM), atomic force microscopy (AFM), and transmission electron microscopy (TEM). Increased amounts of PI103, doxorubicin, and cisplatin were released from CNPs through controlled and continuous manner over 120 h at pH = 5.5 compared to neutral pH. The CNPs showed much enhanced in vitro cytotoxicity in HeLa, HL60, MCF7, and MDA-MB-231 cancer cells compared to a free drug cocktail at 24 and 48 h by inducing apoptosis. Confocal laser scanning microscopy (CLSM) imaging revealed that indeed these CNPs were internalized into subcellular lysosomes through endocytosis in a time dependent mode over 6 h and retained inside for 48 h in HeLa, MDA-MB-231, and MCF7 cells. These CNPs showed their efficacy by damaging DNA and inhibiting Akt as a downstream modulator of PI3K signaling in HeLa cervical cancer cells. These CNPs have the potential to open up new directions in next-generation nanomedicine by simultaneous targeting of multiple oncogenic signaling pathways and inducing DNA damage for augmented therapeutic outcome by reducing toxic side effects and overcoming drug resistance.