Receptor-positive KB Research Articles

Design, characterization and evaluation of a new 99mTc-labeled folate derivative with affinity towards folate receptor

Folate receptors (FRs) are known to be over-expressed in several human malignancies and therefore serve as an important target for small radiolabeled folate derivatives for non-invasive imaging of tumor, which is an important tool for future treatment recourse. In the present article, we report the synthesis of a new 99mTc-labeled radiotracer for the aforementioned application following the well-established 99mTc-‘4+1’ chemistry. Formation of the desired [99mTc]Tc-complex with >95% radiochemical purity was confirmed by radio-HPLC and its structure was ascertained by characterizing a natural rhenium analogue of the said complex. Although the ligand exhibited a weaker affinity towards FRs compared to native folic acid (IC50 8.09 µM vs 29.46 nM), the 99mTc-labeled complex was found to bind folate receptor-positive KB cells with high specificity (∼90%). Similar studies in a folate receptor negative cell line viz. A549 further corroborated the receptor-specificity of the synthesized complex. In vivo studies in KB tumor xenograft showed moderate uptake of ∼2.6% upto 3 h post-injection with high specificity (∼80%). The favorable features observed warrant further screening of the current design towards achieving an improved molecular probe for the said application.

Design and synthesis of a folate-receptor targeted diazepine-ring-opened pyrrolobenzodiazepine prodrug conjugate

Pyrrolobenzodiazepines (PBDs) and their dimers (bis-PBDs) have emerged as some of the most potent chemotherapeutic compounds and are currently under development as novel payloads in antibody-drug conjugates (ADCs). However, when used as stand-alone therapeutics or as warheads for small molecule drug conjugates (SMDCs), dose-limiting toxicities are often observed. As an elegant solution to this inherent problem, we designed and synthesized a diazepine-ring-opened bis-PBD prodrug (pro-PBD-PBD) folate conjugate lacking the one of the two imine moieties found in the corresponding free bis-PBD. Upon entering a targeted cell, cleavage of the linker system, including the hydrolysis of an oxazolidine moiety, results in the formation of a reactive intermediate which possesses a newly formed aldehyde as well as an aromatic amine. A fast and spontaneous intramolecular ring-closing reaction subsequently takes place as the aromatic amine adds to the aldehyde with the loss of water to give the imine, and as a result, the diazepine ring, thereby delivering the bis-PBD to the targeted cell. The in vitro and in vivo activity of this conjugate has been evaluated on folate receptor positive KB cells. Sub-nanomolar activity with good specificity and high cure rates with minimal toxicity have been observed.

Folate-Targeted mRNA Delivery Using Chitosan-Functionalized Selenium Nanoparticles: Potential in Cancer Immunotherapy.

Systemic messenger RNA (mRNA) delivery, although still in its infancy, holds immense potential for application in cancer vaccination and immunotherapy. Its advantages over DNA transfection make it attractive in applications where transient expression is desired. However, this has proved challenging due to mRNA’s instability and susceptibility to degradation. Selenium is important for immune function and modulation, with selenium nanoparticles (SeNPs) finding a niche in biomedicine as drug delivery vehicles, owing to their biocompatibility, low toxicity, and biodegradability. In this investigation, we synthesized chitosan-coated SeNPs with a folic acid targeting moiety for Fluc mRNA delivery to cancer cells in vitro. Synthesized SeNPs were stable and well dispersed, and ranged from 59 to 102 nm in size. Nanoparticles bound and protected mRNA from RNase degradation, while exhibiting low cytotoxicity in the human embryonic kidney (HEK293), breast adenocarcinoma (MCF-7), and nasopharyngeal (KB) cells in culture. Moderate cytotoxicity evidenced in the colorectal carcinoma (Caco-2) and colon carcinoma (HT-29) cells was attributed to apoptosis induction by selenium, as confirmed by acridine orange/ethidium bromide staining. Selenium uptake studies corroborated the transfection results, where significant transgene expression was evident for the overexpressed folate receptor-positive KB cells when compared to the other cells with less or no folate receptors.

Core‐Shell Functionalized Zirconium‐Pemetrexed Coordination Nanoparticles as Carriers with a High Drug Content

AbstractSelected drug molecules with Lewis base functions can be assembled into coordinative nanoparticles (NPs) by linking them with suitable metal ions. Such nanomaterials exhibit a high material economy due to high drug contents and minor amounts of inactive additives. The antifolate pemetrexed (PMX) which is used for the treatment of lung cancers contains two carboxy functions that are able to undergo coordinative binding of metal ions. This study presents the development of a multilayer PMX NP system where each layer serves a distinct purpose. The metal‐drug NP core is assembled in a bottom‐up approach by coordinative interactions between zirconium (IV) ions and PMX molecules. Since the NP core is generated from drug molecules as essential units, it features a very high drug content of almost 80%. The NP core is stabilized against serum with a shell of a polymerized oligoamine‐modified trimethoxysilane derivative (TMSP). As external layer, a polyglutamate‐block‐polysarcosine‐N3 (pGlu‐b‐pSar) coating mediates efficient colloidal stabilization and enables introduction of targeting functionalities by click chemistry. Attaching folate or transferrin ligands to the polymer layer enhances NP uptake into target receptor positive KB and L1210 cells. This study illustrates the development and characterization of metal‐drug coordination NPs with high drug content and variable external functionalizations.

Synthesis of 68Ga-Labeled Biopolymer-based Nanoparticle Imaging Agents for Positron-emission Tomography.

The purpose of this study was to develop a folate receptor-targeted 68Ga-labeled agent for the detection of cancer cells in mouse models of ovarian cancer by dual positron-emission tomography (PET) and magnetic resonance imaging (MRI). Moreover, we aimed to develop a controlled biopolymer-based chemistry that enables linking metal-binding (here Ga-68) chelators. The nanoparticle (NP) agent was created by self-assembling of folic acid-modified polyglutamic acid and chelator-modified chitosan followed by radiolabeling with 68Ga (III) ions (68Ga-NODAGA-FA). The structure of modified biopolymers was characterized by spectroscopy. Particle size and mobility were determined. Significant selective binding of NPs was established in vitro using folate receptor-positive KB and - negative MDA-MB-231 cell lines. In vivo tumor uptake of folate-targeted 68Ga3+-radiolabeled NPs was tested using subcutaneous tumor-bearing CB17 SCID mice models. PET/MR dual modalities showed high tumor uptake with 6.5 tumor-to-muscle ratio and NP localization. In vivo results supporting the preliminary in vitro tests demonstrated considerably higher 68Ga-NODAGA-FA nanoparticle accumulation in KB tumors than in MDA-MB-231 tumors, thereby confirming the folate receptor-mediated uptake of this novel potential PET imaging agent.

The targeting ability of fluorescent quantum dots to the folate receptor rich tumors.

Quantum dots (QDs) bring new insights in cancer theranostics. Exceptional brightness together with the simple possibility to modify surface with targeting molecules make QDs attractive agents in fluorescence guided surgery and photodynamic therapy. Currently, many targeted QDs have been developed for theranostic purpose. However, their targeting ability was tested mainly in two dimensional monolayer tumor cell models, while our study includes 3D tumor model reflecting the specificity of in vivo tumor environment. Core/multilayer shell CdSe/CdS/ZnS QDs were conjugated with folic acid (FA) and characterized spectroscopically. Cytotoxicity of QDs on KB and A549 cells lines were evaluated using the MTT assay. Cellular uptake of QDs was assessed by epifluorescent microscopy. To study the distribution of QDs in tumor tissue, KB spheroids were prepared by means of the liquid overlay technique and then frozen cut of spheroids treated with QDs were imaged by epifluorescence microscopy. We confirmed the specificity of QD-FA for the folic acid receptor positive KB cells. In 3D tumor spheroid model we demonstrated uptake enhancement of QD-FA compared with non-targeted QD. It was demonstrated that penetration profiles were similar for both QDs with penetration depth never exceeding 100 μm. We have demonstrated the effectiveness of FA conjugated QDs to target tumor spheroids thus confirming the crucial role of FRα receptor as a target. Further improvement of QD-FA targeting ability could be performed using dual targeting different targeting agents, such as FA and cyclic RGD.

Targeting of folate-conjugated liposomes with co-entrapped drugs to prostate cancer cells via prostate-specific membrane antigen (PSMA)

Folate-targeted liposomes (FTL) were tested as drug delivery vehicles to PSMA-positive cancer cells. We used FL with co-entrapped mitomycin C lipophilic prodrug (MLP) and doxorubicin (DOX), and the LNCaP prostate cancer cell line which expresses PSMA but is negative for folate receptor. A major increase in cell drug levels was observed when LNCaP cells were incubated with FTL as compared to non-targeted liposomes (NTL). MLP was activated to mitomycin C, and intracellular and nuclear fluorescence of DOX was detected, indicating FTL processing and drug bioavailability. PMPA (2-(phosphonomethyl)-pentanedioic acid), a specific inhibitor of PSMA, blocked the uptake of FTL into LNCaP cells, but did not affect the uptake of FTL into PSMA-deficient and folate receptor-positive KB cells. The cytotoxic activity of drug-loaded FTL was found significantly enhanced when compared to NTL in LNCaP cells. FTL may provide a new tool for targeted therapy of cancers that over-express the PSMA receptor.

Preparation of folate-NOTA-Al18F targeting folate receptor and its microPET/CT imaging

Objective To prepare PET molecular probe folate-NOTA-Al18F(18F-FNA) and to explore its feasibility as an imaging agent in folate receptor positive KB tumor. Methods 18F-FNA was prepared by the method of aluminum fluoride coordination labeling, and the effect of phase transfer catalyst K2.2.2 on the labeling yield was evaluated. Biological distribution was carried out at 10, 30 and 90 min after injection of 3.7 MBq 18F-FNA in nude mice (n=20) xenografted with KB tumor, and the radioactive uptake (%ID/g) and T/NT ratios were then calculated in different organs or tissues. MicroPET imaging was performed at 40 min after injection of 18F-FNA (3.7 MBq). Results The labeling yield of 18F-FNA increased with the presence of K2.2.2. The radiochemical yield was above 98%. The radiochemical purity was above 99%, and still above 98% after maintained in PBS and FBS at 37 ℃ for 4 h. The biodistribution showed that the blood clearance of the probe was slow, and the uptakes in kidneys and tumor which overexpressed folate receptor were significantly high ((5.12±0.58) %ID/g and (1.37±0.20) %ID/g). The high radioactive uptake was observed in KB xenografted mice using microPET imaging. Conclusions The labeling yield of 18F-FNA could increase with the presence of K2.2.2. Furthermore, the encouraging biological distribution and microPET imaging results indicate that 18F-FNA may be a candidate for PET imaging in targeting folate receptor. Key words: Molecular probes; Folic acid; Fluorine radioisotopes; Positron-emission tomography; Tomography, X-ray computed; Mice, nude

Toward Artificial Immunotoxins: Traceless Reversible Conjugation of RNase A with Receptor Targeting and Endosomal Escape Domains.

The specific transport of bioactive proteins into designated target cells is an interesting and challenging perspective for the generation of innovative biopharmaceuticals. Natural protein cytotoxins perform this task with outstanding efficacy. They enter cells with receptor-targeted specificity, respond to changing intracellular microenvironments, and by various mechanisms translocate their cytotoxic protein subunit into the cytosol. Here we imitate this toxin-based delivery strategy in an artificial setting, by bioreversible conjugation of a cytotoxic cargo protein (RNase A) with receptor-targeting PEG-folate and the pH-specific endosomolytic peptide INF7 as synthetic delivery domains. Covalent modification of the cargo protein was achieved using the pH-labile AzMMMan linker and copper-free click chemistry with DBCO-modified delivery modules. This linkage is supposed to enable traceless intracellular release of the RNase A after exposure to the endosomal weakly acidic environment. Delivery of RNase A via this polycation-free delivery strategy resulted in high cytotoxicity against receptor-positive KB tumor cells only when both PEG-folate and INF7 were attached.

Ligand-targeted bacterial minicells: Futuristic nano-sized drug delivery system for the efficient and cost effective delivery of shRNA to cancer cells

In this study, shRNA against VEGFA was packaged in bacterial minicells and surface of minicells was modified with folic acid. Analysis of cellular internalization revealed that folic acid conjugated minicells internalized through receptor mediated endocytosis in folate and PSMA receptor positive KB and LNCaP cells, respectively. In contrast, A549 (folate receptor negative) cells showed minute internalization. In vitro delivery of FAminicellsVEGFA significantly reduced the expression of VEGFA mRNA in KB and LNCaP cells whereas expression of VEGFA remained unaltered in A549 cells. FAminicellsVEGFA significantly reduced tumor volume in mice bearing KB and LNCaP xenograft. On contrary, gradual increase in the tumor volume was recorded in A549 xenograft. FAminicellsVEGFA significantly silenced the VEGFA mRNA in KB and LNCaP xenograft. Expression of VEGFA remained same in FAminicellsVEGFA delivered A549 xenograft. In vivo biodistribution study showed that majority of FAminicellsVEGFA were localized in the tumor followed by intravenous administration.

Promising prospects for 44Sc-/47Sc-based theragnostics: application of 47Sc for radionuclide tumor therapy in mice.

In recent years, (47)Sc has attracted attention because of its favorable decay characteristics (half-life, 3.35 d; average energy, 162 keV; Eγ, 159 keV) for therapeutic application and for SPECT imaging. The aim of the present study was to investigate the suitability of (47)Sc for radionuclide therapy in a preclinical setting. For this purpose a novel DOTA-folate conjugate (cm10) with an albumin-binding entity was used. (47)Sc was produced via the (46)Ca(n,γ)(47)Ca[Formula: see text](47)Sc nuclear reaction at the high-flux reactor at the Institut Laue-Langevin. Separation of the (47)Sc from the target material was performed by a semi-automated process using extraction chromatography and cation exchange chromatography. (47)Sc-labeled cm10 was tested on folate receptor-positive KB tumor cells in vitro. Biodistribution and SPECT imaging experiments were performed in KB tumor-bearing mice. Radionuclide therapy was conducted with two groups of mice, which received either (47)Sc-cm10 (10 MBq) or only saline. Tumor growth and survival time were compared between the two groups of mice. Irradiation of (46)Ca resulted in approximately 1.8 GBq of (47)Ca, which subsequently decayed to (47)Sc. Separation of (47)Sc from (47)Ca was obtained with 80% yield in only 10 min. The (47)Sc was then available in a small volume (∼500 μL) of an ammonium acetate/HCl (pH 4.5) solution suitable for direct radiolabeling. (47)Sc-cm10 was prepared with a radiochemical yield of more than 96% at a specific activity of up to 13 MBq/nmol. In vitro (47)Sc-cm10 showed folate receptor-specific binding and uptake into KB tumor cells. In vivo SPECT/CT images allowed the visualization of accumulated radioactivity in KB tumors and in the kidneys. The therapy study showed a significantly delayed tumor growth in mice, which received (47)Sc-cm10 (10 MBq, 10 Gy) resulting in a more than 50% increase in survival time, compared with untreated control mice. With this study, we demonstrated the suitability of using (47)Sc for therapeutic purposes. On the basis of our recent results obtained with (44)Sc-folate, the present work confirms the applicability of (44)Sc/(47)Sc as an excellent matched pair of nuclides for PET imaging and radionuclide therapy.

Native chemical ligation for conversion of sequence-defined oligomers into targeted pDNA and siRNA carriers

Native chemical ligation (NCL) was established for the conversion of sequence-defined oligomers of different topologies into targeted and PEG shielded pDNA and siRNA carriers. From an existing library of non-targeted oligoethanamino amides, six oligomers containing N-terminal cysteines were selected as cationic cores, to which monodisperse polyethylene glycol (PEG) containing terminal folic acid as targeting ligand (or terminal alanine as targeting negative control ligand) were attached by NCL. Ligated conjugates plus controls (in sum 18 oligomers) were evaluated for pDNA or siRNA gene delivery. Biophysical characteristics including nucleic acid binding in the absence or presence of serum, as well as biological activities in cellular uptake and gene transfer (or gene silencing, respectively) were determined. In most cases, the folic acid-PEG-ligated oligomers displayed a strongly improved cellular binding, uptake and gene transfer into receptor-positive KB cells as compared to the alanine-PEG controls. Changing the topological structures by increasing the number of cationic arms, adding tyrosine trimers as polyplex stabilizing domains, or histidines facilitating endosomal escape resulted in beneficial gene transfer characteristics. The screen revealed different requirements for pDNA and siRNA delivery. A folate-PEG ligated histidinylated four-arm oligomer was most effective for pDNA delivery but inactive for siRNA, whereas a folate-PEG-ligated three-arm oligomer with tyrosine trimer modifications was most effective in siRNA mediated gene silencing. The results demonstrate the site-selective NCL reaction as powerful method to modify existing oligomers. Thus multifunctional targeted carriers can be obtained with ease and used to identify lead structures for subsequent in vivo delivery.

Promises of Cyclotron-Produced 44Sc as a Diagnostic Match for Trivalent β−-Emitters: In Vitro and In Vivo Study of a 44Sc-DOTA-Folate Conjugate

In recent years, implementation of (68)Ga-radiometalated peptides for PET imaging of cancer has attracted the attention of clinicians. Herein, we propose the use of (44)Sc (half-life = 3.97 h, average β(+) energy [Eβ(+)av] = 632 keV) as a valuable alternative to (68)Ga (half-life = 68 min, Eβ(+)av = 830 keV) for imaging and dosimetry before (177)Lu-based radionuclide therapy. The aim of the study was the preclinical evaluation of a folate conjugate labeled with cyclotron-produced (44)Sc and its in vitro and in vivo comparison with the (177)Lu-labeled pendant. (44)Sc was produced via the (44)Ca(p,n)(44)Sc nuclear reaction at a cyclotron (17.6 ± 1.8 MeV, 50 μA, 30 min) using an enriched (44)Ca target (10 mg (44)CaCO3, 97.00%). Separation from the target material was performed by a semiautomated process using extraction chromatography and cation exchange chromatography. Radiolabeling of a DOTA-folate conjugate (cm09) was performed at 95°C within 10 min. The stability of (44)Sc-cm09 was tested in human plasma. (44)Sc-cm09 was investigated in vitro using folate receptor-positive KB tumor cells and in vivo by PET/CT imaging of tumor-bearing mice Under the given irradiation conditions, (44)Sc was obtained in a maximum yield of 350 MBq at high radionuclide purity (>99%). Semiautomated isolation of (44)Sc from (44)Ca targets allowed formulation of up to 300 MBq of (44)Sc in a volume of 200-400 μL of ammonium acetate/HCl solution (1 M, pH 3.5-4.0) within 10 min. Radiolabeling of cm09 was achieved with a radiochemical yield of greater than 96% at a specific activity of 5.2 MBq/nmol. In vitro, (44)Sc-cm09 was stable in human plasma over the whole time of investigation and showed folate receptor-specific binding to KB tumor cells. PET/CT images of mice injected with (44)Sc-cm09 allowed excellent visualization of tumor xenografts. Comparison of cm09 labeled with (44)Sc and (177)Lu revealed almost identical pharmacokinetics. This study presents a high-yield production and efficient separation method of (44)Sc at a quality suitable for radiolabeling of DOTA-functionalized biomolecules. An in vivo proof-of-concept study using a DOTA-folate conjugate demonstrated the excellent features of (44)Sc for PET imaging. Thus, (44)Sc is a valid alternative to (68)Ga for imaging and dosimetry before (177)Lu-radionuclide tumor therapy.

Comparative evaluation of the biological properties of reducible and acid-sensitive folate prodrugs of a highly potent doxorubicin derivative

Two new water-soluble folate receptor-targeted drug conjugates that contain the highly active doxorubicin derivative N-(5,5-diacetoxybut-1-yl)doxorubicin were designed and evaluated for their biological activity against folate receptor positive tumours. The prodrugs were designed to contain an acid-sensitive hydrazone bond KO019 or in addition a disulphide bond KO013 in order to elucidate the importance of the pre-determined breaking point for their in vitro and in vivo properties. Fluorescence microscopy studies confirmed higher uptake of the prodrugs in folate receptor positive KB cells than in the folate receptor negative A549 lung cancer cells. In subsequent in vivo studies in the folate receptor positive KB xenograft model, KO019 was as active as the free drug but significantly less toxic when dosed at twice the dose of the free drug whereas KO013 showed no anticancer efficacy. As an explanation, we could show by HPLC that the prodrug KO013 that additionally contains a disulphide bond undergoes rapid disulphide exchange in murine plasma in the order of 40% after 5h at 37°C in contrast to KO019 which was essentially stable after a 5h incubation.

Cytoplasmic Delivery of Liposomal Contents Mediated by an Acid-Labile Cholesterol-Vinyl Ether-PEG Conjugate

An acid-cleavable PEG lipid, 1'-(4'-cholesteryloxy-3'-butenyl)-omega-methoxy-polyethylene[112] glycolate (CVEP), has been developed that produces stable liposomes when dispersed as a minor component (0.5-5 mol %) in 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). Cleavage of CVEP at mildly acidic pHs results in dePEGylation of the latently fusogenic DOPE liposomes, thereby triggering the onset of content release. This paper describes the synthesis of CVEP via a six-step sequence starting from the readily available precursors 1,4-butanediol, cholesterol, and mPEG acid. The hydrolysis rates and release kinetics from CVEP/DOPE liposome dispersions as a function of CVEP loading, as well as the cryogenic transmission electron microscopy and pH-dependent monolayer properties of 9:91 CVEP/DOPE mixtures, also are reported. When folate receptor-positive KB cells were exposed to calcein-loaded 5:95 CVEP/DOPE liposomes containing 0.1 mol % folate-modified 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-polyethylene[76] glycolamide (folate-PEG-DSPE), delivery of the calcein cargo to the cytoplasm of the cells was observed as determined by fluorescence microscopy and flow cytometry. Fluorescence resonance energy transfer analysis of lipid mixing in these cells was consistent with membrane-membrane fusion between the liposome and endosomal membranes.

Design and synthesis of [111In]DTPA-folate for use as a tumor-targeted radiopharmaceutical.

Folate-conjugated metal chelates have been proposed as potential imaging agents for cancers that overexpress folate receptors. In a previous study, folic acid was linked through its gamma-carboxyl group to deferoxamine (DF), and the 67Ga-labeled complex ([67Ga]DF-folate) was examined for in vivo tumor targeting efficiency in athymic mice with a human tumor cell implant. Although superb tumor-to-background contrast was obtained, slow hepatobiliary clearance would compromise imaging of abdominal tumors such as ovarian cancer. In the present study, folic acid was conjugated to an alternative chelator, diethylenetriaminepentaacetic acid (DTPA), via an ethylenediamine spacer. The desired DTPA-folate (gamma) regioisomer was synthesized by two different approaches, purified by reversed phase column chromatography, and characterized mainly by analytical HPLC, mass spectroscopy, and NMR. In cultured tumor cells, uptake of [111In]DTPA-folate (gamma) was found to be specific for folate receptor-bearing cells, and the kinetics of uptake were similar to those of free folate and other folate-conjugated molecules. In the normal rat, intravenously administered [111In]DTPA-folate (gamma) was found to be rapidly excreted into the urine, giving intestinal levels of radiotracer 10-fold lower than those observed with [67Ga]DF-folate (gamma) at 4 h. In a preliminary mouse imaging study, a folate receptor-positive KB cell tumor was readily visualized by gamma scintigraphy 1 h following intravenous administration of [111In]DTPA-folate (gamma).

The fate of ileorectal anastomosis in Crohn's disease.

An assessment has been made of 25 patients who have undergone ileorectal anastomosis for Crohn's disease. The rate of anastomotic leakage was high (32%) but was fatal in only one patient. The likelihood of leakage was not affected by a safety valve ileostomy, failure to excise all diseased bowel, or steroid therapy. The overall recurrence rate was 60 per cent. This was not affected by the age of the patient, duration of the disease, steroid therapy, or anastomotic leak but was affected adversely by the presence of concurrent small bowel disease and by residual rectal disease at the site of the anastomosis.