Lalit N. Goswami

Covalently Dye-Linked, Surface-Controlled, and Bioconjugated Organically Modified Silica Nanoparticles as Targeted Probes for Optical Imaging

In this paper we report the synthesis and characterization of organically modified silica (ORMOSIL) nanoparticles, covalently incorporating the fluorophore rhodamine-B, and surface-functionalized with a variety of active groups. The synthesized nanoparticles are of ultralow size (diameter approximately 20 nm), highly monodispersed, stable in aqueous suspension, and retain the optical properties of the incorporated fluorophore. The surface of the nanoparticles can be functionalized with a variety of active groups such as hydroxyl, thiol, amine, and carboxyl. The carboxyl groups on the surface were used to conjugate with various bioactive molecules such as transferrin, as well as monoclonal antibodies such as anti-claudin 4 and anti-mesothelin, for targeted delivery to pancreatic cancer cell lines. In vitro experiments have revealed that the cellular uptake of these bioconjugated (targeted) nanoparticles is significantly higher than that of the nonconjugated ones. The ease of surface functionalization and incorporation of a variety of biotargeting molecules, combined with their observed noncytotoxicity, makes these fluorescent ORMOSIL nanoparticles potential candidates as efficient probes for optical bioimaging, both in vitro and in vivo.

Novel Methods to Incorporate Photosensitizers Into Nanocarriers for Cancer Treatment by Photodynamic Therapy

A hydrophobic photosensitizer, 2‐[1‐hexyloxyethyl]‐2‐devinyl pyropheophorbide‐a (HPPH), was loaded into nontoxic biodegradable amine functionalized polyacrylamide (AFPAA) nanoparticles using three different methods (encapsulation, conjugation, and post‐loading), forming a stable aqueous dispersion. Each formulation was characterized for physicochemical properties as well as for photodynamic performance so as to determine the most effective nanocarrier formulation containing HPPH for photodynamic therapy (PDT).

Efficient synthesis of diverse heterobifunctionalized clickable oligo(ethylene glycol) linkers: potential applications in bioconjugation and targeted drug delivery

Herein we describe the sequential synthesis of a variety of azide-alkyne click chemistry-compatible heterobifunctional oligo(ethylene glycol) (OEG) linkers for bioconjugation chemistry applications. Synthesis of these bioorthogonal linkers was accomplished through desymmetrization of OEGs by conversion of one of the hydroxyl groups to either an alkyne or azido functionality. The remaining distal hydroxyl group on the OEGs was activated by either a 4-nitrophenyl carbonate or a mesylate (-OMs) group. The -OMs functional group served as a useful precursor to form a variety of heterobifunctionalized OEG linkers containing different highly reactive end groups, e.g., iodo, -NH(2), -SH and maleimido, that were orthogonal to the alkyne or azido functional group. Also, the alkyne- and azide-terminated OEGs are useful for generating larger discrete poly(ethylene glycol) (PEG) linkers (e.g., PEG(16) and PEG(24)) by employing a Cu(I)-catalyzed 1,3-dipolar cycloaddition click reaction. The utility of these clickable heterobifunctional OEGs in bioconjugation chemistry was demonstrated by attachment of the integrin (α(v)β(3)) receptor targeting peptide, cyclo-(Arg-Gly-Asp-D-Phe-Lys) (cRGfKD) and to the fluorescent probe sulfo-rhodamine B. The synthetic methodology presented herein is suitable for the large scale production of several novel heterobifunctionalized OEGs from readily available and inexpensive starting materials.

Discrete nanomolecular polyhedral borane scaffold supporting multiple gadolinium(III) complexes as a high performance MRI contrast agent.

An icosahedral closo-B(12)(2-) scaffold supports 12 copies of Gd(3+)-chelate held in close proximity with each other by suitable linkers which employ azide-alkyne click chemistry. This design is the first member of a new class of polyfunctional MRI contrast agents carrying a high payload of Gd(3+)-chelate in a sterically constrained configuration. The resulting contrast agent shows higher relaxivity values at high magnetic fields. MRI contrast agents currently in use are not as effective in this regard, presumably due to a lack of steric constraint of gadolinium centers and lower water exchange rates. In vivo MRI studies in mice show excellent contrast enhancement even at one-seventh of the safe clinical dose (0.04 mmol Gd/kg) for up to a 1 h exposure.

Photosensitizers Derived from 132-Oxo-methyl Pyropheophorbide-a: Enhanced Effect of Indium(III) as a Central Metal in In Vitro and In Vivo Photosensitizing Efficacy

Abstract The effects of an additional keto group on absorption wavelength and the corresponding metal complexes Zn(II), Cu(II) In(III) on singlet oxygen production and photodynamic efficacy were examined among the alkyl ether analogs of pyropheophorbide-a. For the preparation of the desired photosensitizers, the methyl 132-oxo-pyropheophorbide-a obtained by reacting methyl pyropheophorbide-a with aqueous LiOH-THF was converted into a series of alkyl ether analogs. These compounds were evaluated for photophysical properties and in vitro (by means of the MTT assay and intracellular localization in RIF cells) and in vivo (in C3H mice implanted with RIF tumors) photosensitizing efficacy. Among the alkyl ether derivatives, the methyl 3-decyloxyethyl-3-devinyl-132-oxo-pyropheophorbide-a was found to be most effective and the insertion of In(III) into this analog further enhanced its in vitro and in vivo photosensitizing efficacy. Fluorescence microscopy showed that, in contrast to the hexyl and dodecyl ether derivatives of HPPH (which localize in mitochondria and lysosomes, respectively), the diketo-analogs and their In(III) complexes localized in Golgi bodies. The preliminary in vitro and in vivo results suggest that, in both free-base and metalated analogs, the introduction of an additional keto group at the five-member exocyclic ring in pyropheophorbide-a diminishes its photosensitizing efficacy. This may be due to a shift in subcellular localization from mitochondria to the Golgi bodies. The further introduction of In(III) enhances photoactivity, but not by shifting the localization of the photosensitizer.

Comparative tumor imaging and PDT Efficacy of HPPH conjugated in the mono- and di-forms to various polymethine cyanine dyes: part - 2.

Previous reports from our laboratory have shown that a bifunctional agent obtained by conjugating a photosensitizer (HPPH) to a cyanine dye (CD) can be used for fluorescence image-guided treatment of tumor by photodynamic therapy (PDT). However, the resulting HPPH-CD conjugate showed a significant difference between the tumor-imaging and therapeutic doses. It was demonstrated that the singlet oxygen (1O2*, a key cytotoxic agent in PDT) produced by the conjugate upon excitation of the HPPH moiety was partially quenched by the CD-moiety; this resulted in a reduced PDT response when compared to HPPH-PDT under similar treatment parameters. To improve the therapeutic potential of the conjugate, we synthesized a series of dual functional agents in which one or two HPPH moieties were separately conjugated to three different dyes (Cypate, modified IR820 or modified IR783). The newly synthesized conjugates were compared with our lead compound HPPH-CD in terms of photophysical properties, in vitro and in vivo PDT efficacy, tumor uptake and imaging potential. Among the analogs investigated, the conjugate, in which two HPPH moieties were linked to the modified IR820 produced enhanced tumor uptake and tumor contrast in both Colon 26 (a murine Colon carcinoma) and U87 (a human glioblastoma) cell lines. The long-term PDT efficacy (cure) of this conjugate in BALB/c mice, bearing Colon 26 tumors was also enhanced; however, its efficacy in Nude mice bearing U87 tumors was slightly reduced. It was also found that in all the conjugates the singlet oxygen generation and, consequently, PDT efficacy were compromised by a competing pathway, whereby an electronic excitation of HPPH, the energy donor, is deactivated through an electronic excitation energy transfer (Forster Resonance Energy Transfer, FRET) to the CD fluorophore, the energy acceptor, resulting in overall reduction of the singlet oxygen production. Conjugates with increased FRET showed reduced singlet oxygen production and PDT efficacy. Among the conjugates investigated, the bifunctional agent in which two HPPH moieties were linked to the benzoindole-based cyanine dye 11 showed superiority over the lead candidate 9 (mono HPPH-cyanine dye).

cRGD Peptide-Conjugated Icosahedral closo-B122− Core Carrying Multiple Gd3+-DOTA Chelates for αvβ3 Integrin-Targeted Tumor Imaging (MRI)

A vertex-differentiated icosahedral closo-B(12)(2-) core was utilized to construct a α(v)β(3) integrin receptor-targeted (via cRGD peptide) high payload MRI contrast agent (CA-12) carrying 11 copies of Gd(3+)-DOTA chelates attached to the closo-B(12)(2-) surface via suitable linkers. The resulting polyfunctional MRI contrast agent possessed a higher relaxivity value per-Gd compared to Omniscan, a small molecular contrast agent commonly used in clinical settings. The α(v)β(3) integrin receptor specificity of CA-12 was confirmed via in vitro cellular binding experiments and in vivo MRI of mice bearing human PC-3 prostate cancer xenografts. Integrin α(v)β(3)-positive MDA-MB-231 cells exhibited 300% higher uptake of CA-12 than α(v)β(3)-negative T47D cells. Serial T1-weighted MRI showed superior contrast enhancement of tumors by CA-12 compared to both a nontargeted 12-fold Gd(3+)-DOTA closomer control (CA-7) and Omniscan. Contrast enhancement by CA-12 persisted for 4 h postinjection, and subsequent enhancement of kidney tissue indicated a renal elimination route similar to Omniscan. No toxic effects of CA-12 were apparent in any mice for up to 24 h postinjection. Post-mortem ICP-OES analysis at 24 h detected no residual Gd in any of the tissue samples analyzed.

Extensions of the Icosahedral Closomer Structure by Using Azide–Alkyne Click Reactions†

Polyhedral boranes and carboranes are of great interest because of their use as a 10B source in BNCT,[1] as hydrophobic pharmacophores,[2] as weakly coordinating anions,[3] and as ligands for transition metals and other types of metal as well.[4] One of the less explored applications of the polyhedral boranes is one in which they can be used as platforms for the targeted and high payload density delivery of drug molecules and imaging agents.[5] The icosahedral dodecahydro-closo-dodecaborate dianion [closo-B12H12]2−, (Figure 1) is an aromatic species having extensive delocalization of thirteen bonding electron pairs[6] as well as unique properties, such as chemical, hydrolytic and thermal stabilities and low toxicity. Each of the twelve vertices present in [closo-B12H12]2− can be attached to either identical or a variety of substituents to generate attractive molecular construction modules. To date the [closo-B12H12]2− cage has been modified with a variety of functional substituents such as hydroxyl,[7] thiol,[8] thioethers,[9] halogens,[10] amines,[11] alkyl, aryl groups and others[12] to form polyhedral boranes with varying degrees of substitution and reactivity. Figure 1 Icosahedral closo-boranes The discovery of the remarkable B-H hydroxylation reaction with H2O2 has led to a variety of polyhedral borane and carborane structures, such as [closo-B12(OH)12]2− (1), [closo-CHB11(OH)11]− and [closo-1,12-C2H2B10(OH)10][13] that have added new dimensions to the chemistry of polyfunctional molecules. The hydroxylation of all of the B-H vertices of [closo-B12H12]2− using 30% hydrogen peroxide provides 1 in greater than 95% yield.[7b] Icosahedral 1 is stable to hydrolysis, air-oxidation and enzymatic attack while providing a functionalized molecular scaffold that can be used to anchor up to twelve radial arms with desired pendant groups even at generation zero. This is possible due to the fact that the reactivity of the B-OH vertices resembles that of alcohols. Consequently, twelve-fold carboxylate ester[14] and ether[15] derivatives, described by us as “closomers”, are now available.[14a] Closomer derivatives of 1 and similar dendrimers share several chracteristics although basic differences are significant. Closomers are the smaller in size with greater rigidity (dendrimers are more loosely constructed while closomers of the same functionality are more rigidly configured as twelve chains which simultaneously originate at the icosahedral surface in close proximity to each other). Closomers have higher symmetry and consist of a single structure. In addition, a much more compact presentation of functional groups is possible with closomer derivatives of 1 as compared to dendrimer structures having similar twelve-fold functionality. Consequently, the chemistry of 1 provides uniform nanoparticle-size molecular architectures potentially useful for carrying payloads of pharmaceuticals, imaging agents or many other useful substitutents. Blending click[16] and closomer chemistries would provide increased opportunities for the syntheses of novel therapeutic and diagnostic entities. One obvious addition of click chemistry to that of the icosahedral borane scaffold requires the initial synthesis of twelve-fold azide-substituted closomers that can react with terminal alkynes to generate twelve-fold 1,2,3-triazole rings. Here we report for the first time several examples of this new chemistry which demonstrates the only known methodology capable of producing twelve reaction centers for the click reaction at generation zero. Below, we extend our original work[14] on the synthesis of ester closomers and describe a method for the synthesis of ester-linked azido closomers by the twelve-fold esterification of (TBA)2-1 [TBA = tetran-butylammonium] with an α-haloacetic anhydride followed by the displacement of halide ion with azide (NaN3). The reaction of TBA2-1 and chloroacetic anhydride (5.0 eqv. per vertex) at the reflux temperature in acetonitrile for 5 days gave the twelve-fold chloroacetate closomer 2 after purification using gelfiltration chromatography on a Lipophilic Sephadex® LH-20 column in 91% yield (Scheme 1). The excess, unreacted chloroacetic anhydride was separated and reutilized. The course of the esterification reactions was monitored by mass spectrometric analysis and 11B NMR spectra of crude reaction mixtures. In the 11B NMR spectrum, intermediate stages of esterification were characterized by an array of peaks centered near −17 ppm. Complete reaction was characterized by equivalent B-OCOR vertices and a singlet near −17 ppm (Fig. SI-1 in supporting information). The 1H NMR of the purified product contained a characteristic singlet at δ 4.1 ppm for 24-protons assigned to twelve-Cl-CH2CO2- groups bound to the substituted closo-borane cage. Scheme 1 Synthetic route for twelve-fold Click reaction with [closo-B12H12]2−; Conditions, a) chloroacetic anhydride in CH3CN, 5 days, reflux; b) NaN3 in DMF, 2 days, 50°C; c) alkyne, CuI, DIPEA, CH3CN-THF (1:1), 2-3 days, RT. DMF = dimethylformamide, ... In a typical procedure for the synthesis of twelve-fold azide terminated closomer 3, closomer 2 was reacted with a 10-fold excess of sodium azide in DMF (dimethylformamide) at 50°C for 2 days to obtain the twelve-fold azido closomer 3 in essentially quantitative yields (Scheme 1). The progress of the reaction was monitored using 1H NMR and mass spectrometric analysis; at this stage the 11B NMR spectrum of the crude reaction mixture had not changed significantly from that of the starting closomer 2. However, in the 1H NMR spectrum, the completion of the reaction was indicated by a complete shift in the characteristic singlet for 24-protons assigned to the twelve Cl-CH2CO2- groups from δ 4.1 ppm to δ 3.7 ppm for the twelve N3-CH2CO2- groups. The IR-spectrum of the product also showed the presence of a characteristic peak at 2109 cm−1 which was attributed to the asymmetrical stretch of the pendant azide-group (Fig. SI-2 in supporting information). Purification of the product was achieved by concentrating the reaction mixture to dryness under reduced pressure, dissolution of the residue in ethyl acetate and filtration using a celite pad to remove unreacted sodium azide and sodium chloride. The filtrate was concentrated to obtain the desired product in quantitative yield. The product could be used in the click reaction with alkyne substrates without further purification. Among various forms of click reactions, the Cu(I)-catalyzed variant of the Huisgen 1,3-dipolar cycloaddition of azides with alkynes to afford 1,2,3-triazoles has emerged as the most popular for click chemistry due to its reliability, specificity and biocompatibility. In a typical click reaction; twelve-fold azido closomer 3 and acetylenic compounds (5 eq. per vertex), in the presence of copper (I) iodide (1 eq. per vertex) and Hunig’s base (10 eq. per vertex) were reacted for 2-3 days under an argon atmosphere (Scheme 1). The progress of the click reaction was followed by mass spectrometric analysis and 1H NMR spectra of crude reaction mixtures. Products were purified by size-exclusion column chromatography (Lipophilic Sephadex® LH-20) using acetonitrile as an eluent to yield click products in good to excellent yields (Table 1). All of the click products showed a characteristic singlet between δ 7-8 ppm for twelve protons of alkene-CH of the twelve triazole rings in the 1H NMR spectrum of the purified products. The IR-spectrum of the product also showed the disappearance of the characteristic peak at 2109 cm−1 which was originally attributed to the asymmetric stretching of the azide-group. Table 1 Click reactions of various terminal alkynes with twelve-fold azido closomer 3 using CuI and DIPEA in CH3CN-THF (1:1). In conclusion, we have established a mild and highly efficient protocol for the synthesis of twelve-chloroacetate esters of (TBA)2-1. The twelve α-chlorine atoms on the ester closomer 2 were readily replaced by an azide functionality, thereby forming a closomer having twelve linker arms with terminal groups, 3, available for the twelve-fold click reaction on a closomer surface. These unique products discrete nano-size molecules carrying multiple copies of diverse functions which serve as model therapeutic and/or diagnostic agents. In addition, the α-chloro and α-azido substituted ester analogues (2 and 3) can be used to perform reactions characteristic of simple alkyl halides and azides, respectively, further expanding the chemical horizons of closo-borane scaffolds. Applications of Huisgen chemistry have expanded the scope of organic chemistry and at this point we can imagine the potential for closo-borane chemistry amplified with click chemistry.

Regioselective Synthesis and Photophysical and Electrochemical Studies of 20‐Substituted Cyanine Dye–Purpurinimide Conjugates: Incorporation of NiII into the Conjugate Enhances its Tumor‐Uptake and Fluorescence‐Imaging Ability

We report herein a simple and efficient approach to the synthesis of a variety of meso-substituted purpurinimides. The reaction of meso-substituted purpurinimide with N-bromosuccinimide regioselectively introduced a bromo functionality at the 20-position, which on further reaction with a variety of boronic acids under Suzuki reaction conditions yielded the corresponding meso-substituted analogues. Interestingly, the free base and the metalated analogues showed remarkable differences in photosensitizing efficacy (PDT) and tumor-imaging ability. For example, the free-base conjugate showed significant in vitro PDT efficacy, but limited tumor avidity in mice bearing tumors, whereas the corresponding Ni(II) derivative did not produce any cell kill, but showed excellent tumor-imaging ability at a dose of 0.3 μmol kg(-1) at 24, 48, and 72 h post-injection. The limited PDT efficacy of the Ni(II) analogue could be due to its inability to produce singlet oxygen, a key cytotoxic agent required for cell kill in PDT. Based on electrochemical and spectroelectrochemical data in DMSO, the first one-electron oxidation (0.52 V vs. SCE) and the first one-electron reduction (-0.57-0.67 V vs. SCE) of both the free base and the corresponding Ni(II) conjugates are centered on the cyanine dye, whereas the second one-electron reduction (-0.81 V vs. SCE) of the two conjugates is assigned to the purpurinimide part of the molecule. Reduction of the cyanine dye unit is facile and occurs prior to reduction of the purpurinimide group, which suggests that the cyanine dye unit as an oxidant could be the driving force for quenching of the excited triplet state of the molecules. An interaction between the cyanine dye and the purpurinimide group is clearly observed in the free-base conjugate, which compares with a negligible interaction between the two functional groups in the Ni(II) conjugate. As a result, the larger HOMO-LUMO gap of the free-base conjugate and the corresponding smaller quenching constant is a reason to decrease the intramolecular quenching process and increase the production of singlet oxygen to some degree.

Synthesis of vertex-differentiated icosahedral closo -boranes: polyfunctional scaffolds for targeted drug delivery

We report methods for the synthesis of vertex-differentiated icosahedral closo-boranes. A single B-OH vertex of the icosahedral borane [closo-B(12)(OH)(12)](2-) was derivatized to prepare [closo-B(12)(OR)(OH)(11)](2-) using optimized alkylation conditions and purification procedures. Several representative vertex-differentiated icosahedral closo-boranes were prepared utilizing carbonate ester and azide-alkyne click chemistries on the surface of the closo-B(12)(2-) core.