Enhancing photodynamic and radionuclide therapy by small interfering RNA (siRNA)-RAD51 transfection via self-emulsifying delivery systems (SNEDDS).

BACKGROUND AIMS: Gene-silencing by small interfering RNA (siRNA) is an attractive therapy to regulate cancer death, tumor recurrence or metastasis. Because siRNAs are easily degraded, it is necessary to develop transport and delivery systems to achieve efficient tumor targeting. Self-nanoemulsifying systems (SNEDDS) have been successfully used for pDNA transport and delivery, so they may be useful for siRNA. The aim of this work is to introduce siRNA-RAD51 into a SNEDDS prepared with Phospholipon-90G, Labrafil-M1944-CS and Cremophor-RH40 and evaluate its efficacy in preventing homologous recombination of DNA double-strand breaks caused by photodynamic therapy (PDT) and ionizing radiation (IR). METHODS: The siRNA-RAD51 was loaded into SNEDDS using chitosan. Transfection capacity was estimated by comparison with Lipofectamine-2000. RESULTS: SNEDDS(siRNA-RAD51) induced gene silencing effect on the therapies evaluated by cell viability and clonogenic assays using T47D breast cancer cells. CONCLUSIONS: SNEDDS(siRNA-RAD51) shown to be an effective siRNA-delivery system to decrease cellular resistance in PDT or IR.

68Ga-DOTA-D-Alanine-BoroPro Radiotracer for Imaging of the Fibroblast Activation Protein in Malignant and Non-Malignant Diseases.

Recently, we reported a new fibroblast activation protein (FAP) inhibitor radiopharmaceutical based on the 99mTc-((R)-1-((6-hydrazinylnicotinoyl)-D-alanyl) pyrrolidin-2-yl) boronic acid (99mTc-HYNIC-D-Alanine-BoroPro)(99mTc-HYNIC-iFAP) structure for tumor microenvironment SPECT imaging. This research aimed to synthesize 68Ga-[2,2',2″,2‴-(2-(4-(2-(5-(((S)-1-((S)-2-boronopyrrolidin-1-yl)-1-oxopropan-2-yl)carbamoyl)pyridin-2-yl)hydrazine-1-carbothioamido)benzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid] (68Ga-DOTA-D-Alanine-BoroPro)(68Ga-iFAP) as a novel radiotracer for PET imaging and evaluate its usefulness for FAP expression in malignant and non-malignant tissues. The coupling of p-SCN-benzene DOTA with HYNIC-iFAP was used for the chemical synthesis and further labeling with 68Ga. Radiochemical purity was verified by radio-HPLC. The specificity of 68Ga-iFAP was evaluated in HCT116 cells, in which FAP expression was verified by immunofluorescence and Western blot. Biodistribution and biokinetic studies were performed in murine models. 68Ga-iFAP uptake at the myocardial level was assessed in mice with induced infarction. First-in-human images of 68Ga-iFAP in healthy subjects and patients with myocardial infarction, glioblastoma, prostate cancer, and breast cancer were also obtained. DOTA-D-Alanine BoroPro was prepared with a chemical purity of 98% and was characterized by UPLC mass spectroscopy, FT-IR, and UV-vis. The 68Ga-iFAP was obtained with a radiochemical purity of >95%. In vitro and in vivo studies demonstrated 68Ga-iFAP-specific recognition for FAP, rapid renal elimination, and adequate visualization of the glioblastoma, breast tumor, prostate cancer, and myocardial infarction sites. The results of this research justify further dosimetry and clinical trials to establish the specificity and sensitivity of 68Ga-iFAP PET for FAP expression imaging.

Pharmaceutical Nanoplatforms Based on Self-nanoemulsifying Drug Delivery Systems for Optimal Transport and Co-delivery of siRNAs and Anticancer Drugs.

Small interfering RNAs (siRNAs) have the ability to induce selective gene silencing, although siRNAs are vulnerable to degradation in vivo. Various active pharmaceutical ingredients (APIs) are currently used as effective therapeutics in the treatment of cancer. However, routes of administration are limited due to their physicochemical and biopharmaceutical properties. This research aimed to develop oral pharmaceutical formulations based on self-nanoemulsifying drug delivery systems (SNEDDS) for optimal transport and co-delivery of siRNAs related to cancer and APIs. Formulations were developed using optimal mixing design (Design-Expert 11 software) for SNEDDS loading with siRNA (water/oil emulsion), API (oil/water emulsion), and siRNA-API (multiphase water/oil/water emulsion). The final formulations were characterized physicochemically and biologically. The nanosystems less than 50 nm in size had a drug loading above 48 %. The highest drug release occurred at intestinal pH, allowing drug protection in physiological fluids. SNEDDS-siRNA-APIs showed a twofold toxicity effect than APIs in solution and higher transfection and internalization of siRNA in cancer cells with respect to free siRNAs. In the duodenum, higher permeability was observed with SNEDDS-API than with the API solution, as determined by ex-vivo fluorescence microscopy. The multifunctional formulation based on SNEDDS was successfully prepared, siRNA, hydrophobic chemotherapeutics (doxorubicin, valrubicin and methotrexate) and photosensitizers (rhodamine b and protoporphyrin IX) agents were loaded, using a chitosan-RNA core, and Labrafil® M 1944 CS, Cremophor® RH40, phosphatidylcholine shell, forming stable hybrid SNEDDS as multiphasic emulsion, suitable as co-delivery system with a potent anticancer activity.

Toxicity Assessment of [177Lu]Lu-iFAP/iPSMA Nanoparticles Prepared under GMP-Compliant Radiopharmaceutical Processes.

The fibroblast activation protein (FAP) is heavily expressed in fibroblasts associated with the tumor microenvironment, while the prostate-specific membrane antigen (PSMA) is expressed in the neovasculature of malignant angiogenic processes. Previously, we reported that [177Lu]lutetium sesquioxide-iFAP/iPSMA nanoparticles ([177Lu]Lu-iFAP/iPSMA) inhibit HCT116 tumor progression in mice. Understanding the toxicity of [177Lu]Lu-iFAP/iPSMA in healthy tissues, as well as at the tissue and cellular level in pathological settings, is essential to demonstrate the nanosystem safety for treating patients. It is equally important to demonstrate that [177Lu]Lu-iFAP/iPSMA can be prepared under good manufacturing practices (GMP) with reproducible pharmaceutical-grade quality characteristics. This research aimed to prepare [177Lu]Lu-iFAP/iPSMA under GMP-compliant radiopharmaceutical processes and evaluate its toxicity in cell cultures and murine biological systems under pathological environments. [177Lu]Lu2O3 nanoparticles were formulated as radiocolloidal solutions with FAP and PSMA inhibitor ligands (iFAP and iPSMA), sodium citrate, and gelatin, followed by heating at 121 °C (103-kPa pressure) for 15 min. Three consecutive batches were manufactured. The final product was analyzed according to conventional pharmacopeial methods. The Lu content in the formulations was determined by X-ray fluorescence. [177Lu]Lu-iFAP/iPSMA performance in cancer cells was evaluated in vitro by immunofluorescence. Histopathological toxicity in healthy and tumor tissues was assessed in HCT116 tumor-bearing mice. Immunohistochemical assays were performed to corroborate FAP and PSMA tumor expression. Acute genotoxicity was evaluated using the micronuclei assay. The results showed that the batches manufactured under GMP conditions were reproducible. Radiocolloidal solutions were sterile and free of bacterial endotoxins, with radionuclidic and radiochemical purity greater than 99%. The lutetium content was 0.10 ± 0.02 mg/mL (0.9 GBq/mg). Significant inhibition of cell proliferation in vitro and in tumors was observed due to the accumulation of nanoparticles in the fibroblasts (FAP+) and neovasculature (PSMA+) of the tumor microenvironment. No histopathological damage was detected in healthy tissues. The data obtained in this research provide new evidence on the selective toxicity to malignant tumors and the absence of histological changes in healthy tissues after intravenous injection of [177Lu]Lu-iFAP/iPSMA in mammalian hosts. The easy preparation under GMP conditions and the toxicity features provide the added value needed for [177Lu]Lu-iFAP/iPSMA clinical translation.

Engineered rHDL Nanoparticles as a Suitable Platform for Theranostic Applications.

Reconstituted high-density lipoproteins (rHDLs) can transport and specifically release drugs and imaging agents, mediated by the Scavenger Receptor Type B1 (SR-B1) present in a wide variety of tumor cells, providing convenient platforms for developing theranostic systems. Usually, phospholipids or Apo-A1 lipoproteins on the particle surfaces are the motifs used to conjugate molecules for the multifunctional purposes of the rHDL nanoparticles. Cholesterol has been less addressed as a region to bind molecules or functional groups to the rHDL surface. To maximize the efficacy and improve the radiolabeling of rHDL theranostic systems, we synthesized compounds with bifunctional agents covalently linked to cholesterol. This strategy means that the radionuclide was bound to the surface, while the therapeutic agent was encapsulated in the lipophilic core. In this research, HYNIC-S-(CH2)3-S-Cholesterol and DOTA-benzene-p-SC-NH-(CH2)2-NH-Cholesterol derivatives were synthesized to prepare nanoparticles (NPs) of HYNIC-rHDL and DOTA-rHDL, which can subsequently be linked to radionuclides for SPECT/PET imaging or targeted radiotherapy. HYNIC is used to complexing 99mTc and DOTA for labeling molecules with 111, 113mIn, 67, 68Ga, 177Lu, 161Tb, 225Ac, and 64Cu, among others. In vitro studies showed that the NPs of HYNIC-rHDL and DOTA-rHDL maintain specific recognition by SR-B1 and the ability to internalize and release, in the cytosol of cancer cells, the molecules carried in their core. The biodistribution in mice showed a similar behavior between rHDL (without surface modification) and HYNIC-rHDL, while DOTA-rHDL exhibited a different biodistribution pattern due to the significant reduction in the lipophilicity of the modified cholesterol molecule. Both systems demonstrated characteristics for the development of suitable theranostic platforms for personalized cancer treatment.

Photoactivation of Chemotherapeutic Agents with Cerenkov Radiation for Chemo-Photodynamic Therapy.

Cerenkov radiation (CR) can be used as an internal light source in photodynamic therapy (PDT). Methotrexate (MTX) and paclitaxel (PTX), chemotherapeutic agents with wide clinical use, have characteristics of photosensitizers (PS). This work evaluates the possibility of photoexciting MTX and PTX with CR from 18F-FDG to produce reactive oxygen species (ROS) capable of inducing cytotoxicity. PTX did not produce ROS when excited by CR from 18F-FDG, so it is not useful for PDT. In contrast, MTX produces 1O2 (detected by ABMA) in amounts sufficient to significantly decrease the viability of the T47D cells. MTX solutions of 100 nM combined with 18F-FDG activities of 50 (1.85 MBq) and 100 µCi (3.7 MBq) produced a significant decrease in cell viability to (50.09 ± 4.95) and (47.96 ± 11.19)%, respectively, compared to MTX (66.29 ± 5.92)% and 18F-FDG (91.35 ± 7.00% for 50 µCi and 99.43 ± 11.03% for 100 µCi) alone. Using the CellRox Green reagent, the intracellular production of ROS was confirmed as the main mechanism of cytotoxicity. The results confirm the therapeutic potential of photoactivation with CR and the synergy of the combined treatment with chemotherapy + photodynamic therapy (CMT + PDT). The combination of chemotherapeutic agents with PS properties and ß-emitting radiopharmaceuticals, previously approved for clinical use, will make it possible to shorten the evaluation stages of new CMT + PDT systems.

Preclinical evaluation of early multi-organ toxicity induced by liposomal doxorubicin using 67Ga-citrate.

Although liposomal doxorubicin (LPD) is widely used for cancer treatment, knowledge concerning the toxicity induced by this drug in healthy organs and tissues is limited. LPD-induced toxicity studies relative to free doxorubicin (DOX) have focused on cardiotoxicity in tumor-bearing animals. On the other hand, the results on DOX-induced cardiotoxicity depending on gender are controversial. One of the manifestations of toxicity is tissue inflammation. 67Ga-citrate has been used for decades to assess inflammation in various pathologies. In this work, the ex vivo biodistribution of 67Ga-citrate is used to evaluate induced multi-organ toxicity in healthy 10-week-old male and female CD1 mice treated for 5 weeks with LPD. Toxicity in males, determined by 67Ga-citrate, was evident only in the target organs of liposomes (spleen, liver, kidneys, and lungs); the average weight loss was 11% and mortality was 14%. In female mice, 67Ga-citrate revealed a cytotoxic effect in practically all organs, the average weight loss was 37%, and the mortality after the last dose of LPD was 66%. These results confirm the usefulness of 67Ga-citrate and the importance of stratifying by sex in the toxicological evaluation of drugs.

225Ac-rHDL Nanoparticles: A Potential Agent for Targeted Alpha-Particle Therapy of Tumors Overexpressing SR-BI Proteins.

Actinium-225 and other alpha-particle-emitting radionuclides have shown high potential for cancer treatment. Reconstituted high-density lipoproteins (rHDL) specifically recognize the scavenger receptor B type I (SR-BI) overexpressed in several types of cancer cells. Furthermore, after rHDL-SR-BI recognition, the rHDL content is injected into the cell cytoplasm. This research aimed to prepare a targeted 225Ac-delivering nanosystem by encapsulating the radionuclide into rHDL nanoparticles. The synthesis of rHDL was performed in two steps using the microfluidic synthesis method for the subsequent encapsulation of 225Ac, previously complexed to a lipophilic molecule (225Ac-DOTA-benzene-p-SCN, CLog P = 3.42). The nanosystem (13 nm particle size) showed a radiochemical purity higher than 99% and stability in human serum. In vitro studies in HEP-G2 and PC-3 cancer cells (SR-BI positive) demonstrated that 225Ac was successfully internalized into the cytoplasm of cells, delivering high radiation doses to cell nuclei (107 Gy to PC-3 and 161 Gy to HEP-G2 nuclei at 24 h), resulting in a significant decrease in cell viability down to 3.22 ± 0.72% for the PC-3 and to 1.79 ± 0.23% for HEP-G2 at 192 h after 225Ac-rHDL treatment. After intratumoral 225Ac-rHDL administration in mice bearing HEP-G2 tumors, the biokinetic profile showed significant retention of radioactivity in the tumor masses (90.16 ± 2.52% of the injected activity), which generated ablative radiation doses (649 Gy/MBq). The results demonstrated adequate properties of rHDL as a stable carrier for selective deposition of 225Ac within cancer cells overexpressing SR-BI. The results obtained in this research justify further preclinical studies, designed to evaluate the therapeutic efficacy of the 225Ac-rHDL system for targeted alpha-particle therapy of tumors that overexpress the SR-BI receptor.

Design, Synthesis and Preclinical Assessment of 99mTc-iFAP for In Vivo Fibroblast Activation Protein (FAP) Imaging.

Fibroblast activation protein (FAP) is expressed in the microenvironment of most human epithelial tumors. 68Ga-labeled FAP inhibitors based on the cyanopyrrolidine structure (FAPI) are currently used for the detection of the tumor microenvironment by PET imaging. This research aimed to design, synthesize and preclinically evaluate a new FAP inhibitor radiopharmaceutical based on the 99mTc-((R)-1-((6-hydrazinylnicotinoyl)-D-alanyl) pyrrolidin-2-yl) boronic acid (99mTc-iFAP) structure for SPECT imaging. Molecular docking for affinity calculations was performed using the AutoDock software. The chemical synthesis was based on a series of coupling reactions of 6-hidrazinylnicotinic acid (HYNIC) and D-alanine to a boronic acid derivative. The iFAP was prepared as a lyophilized formulation based on EDDA/SnCl2 for labeling with 99mTc. The radiochemical purity (R.P.) was verified via ITLC-SG and reversed-phase radio-HPLC. The stability in human serum was evaluated by size-exclusion HPLC. In vitro cell uptake was assessed using N30 stromal endometrial cells (FAP positive) and human fibroblasts (FAP negative). Biodistribution and tumor uptake were determined in Hep-G2 tumor-bearing nude mice, from which images were acquired using a micro-SPECT/CT. The iFAP ligand (Ki = 0.536 nm, AutoDock affinity), characterized by UV-Vis, FT-IR, 1H-NMR and UPLC-mass spectroscopies, was synthesized with a chemical purity of 92%. The 99mTc-iFAP was obtained with a R.P. >98%. In vitro and in vivo studies indicated high radiotracer stability in human serum (>95% at 24 h), specific recognition for FAP, high tumor uptake (7.05 ± 1.13% ID/g at 30 min) and fast kidney elimination. The results found in this research justify additional dosimetric and clinical studies to establish the sensitivity and specificity of the 99mTc-iFAP.

Targeted photodynamic therapy using reconstituted high-density lipoproteins as rhodamine transporters.

Reconstituted high-density lipoprotein (rHDL) nanoparticles are excellent transporters of molecules and very useful for targeted therapy as they specifically recognize the scavenger receptor, class B1 (SR-B1) that is present on the surface of a wide range of tumor cells. However, they have rarely been employed to transport photosensitizers (PS) for photodynamic therapy (PDT). Rhodamine (R) compounds have been dismissed as useful PSs for PDT due to their low 1O2 production, excitation wavelengths with little tissue penetration, and poor selectivity for tumor cells. It was recently demonstrated that when irradiating at 532 nm or with Cerenkov radiation (CR) from a ß-emitting radionuclide, R123, R6G, and RB undergo electron transfer reactions (type I reaction) with folic acid. R6G also produces type I reactions with O2. In this work, the photodynamic effects of the rHDL-R system were evaluated in vitro. rHDL nanoparticles loaded with R123, R6G, and RB were synthesized, and the PS was internalized into T47D tumor cells. When cells were irradiated with a 532-nm laser in the presence of an rHDL-R systems, a cytotoxic photodynamic effect was obtained in the order R6G > R123 > RB. In the presence of CR from a 177Lu source, cytotoxicity showed the order R6G > RB > R123. The higher cytotoxicity induced by R6G in both cases corresponds to higher cellular internalization and larger production of type I and II reactions. Thus, in this work, it is proposed that rHDL-R/177Lu system can be applied in theragnostics as a multimodal radiotherapy-PDT-imaging system (imaging by SPECT or Cerenkov) and in hypoxic solid tumors in which external radiation is not effective and 177Lu-CR acts as light source.

A Multimodal Theranostic System Prepared from High-Density Lipoprotein Carrier of Doxorubicin and 177Lu.

Recently, it was demonstrated that doxorubicin (Dox.HCl), a chemotherapeutic agent, could be photoactivated by Cerenkov radiation (CR). The objective of the present work was to develop a multimodal chemotherapy-radiotherapy-photodynamic therapeutic system based on reconstituted high-density lipoprotein (rHDL) loaded with Dox.HCl and 177Lu-DOTA. 177Lu acts as a therapeutic radionuclide and CR source. The system can be visualized by nuclear imaging. Fluorescence microscopy showed that rHDL-Dox specifically recognized cancer cells (T47D) that are positive for SR-B1 receptors. Encapsulated Dox.HCl was released into the cells and produced reactive oxygen species when irradiated with a 450-nm laser (photodynamic effect). The same effect occurred when Dox.HCl was irradiated by 177Lu CR. Through in vitro experiments, it was confirmed that the addition of 177Lu-DOTA to the rHDL-Dox nanosystem did not affect the specific recognition of SR-B1 receptors expressed in cells, or the cellular internalization of 177Lu-DOTA. The toxicity induced by the rHDL-Dox/177Lu nanosystem in cell lines with high (T47D and PC3), poor (H9C2) and almost-zero (human fibroblasts (FB)) expression of SR-B1 was evaluated in vitro and confirmed the synergy of the combined chemotherapy-radiotherapy-photodynamic therapeutic effect; this induced toxicity was proportional to the expression of the SR-B1 receptor on the surface of the cells used. The HDL-Dox/177Lu nanosystem experienced uptake by tumor cells and the liver-both tissues with high expression of SR-B1 receptors-but not by the heart. 177Lu CR offered the possibility of imparting photodynamic therapy where laser light could not reach.

Nanoradiopharmaceuticals Based on Alpha Emitters: Recent Developments for Medical Applications.

The application of nanotechnology in nuclear medicine offers attractive therapeutic opportunities for the treatment of various diseases, including cancer. Indeed, nanoparticles-conjugated targeted alpha-particle therapy (TAT) would be ideal for localized cell killing due to high linear energy transfer and short ranges of alpha emitters. New approaches in radiolabeling are necessary because chemical radiolabeling techniques are rendered sub-optimal due to the presence of recoil energy generated by alpha decay, which causes chemical bonds to break. This review attempts to cover, in a concise fashion, various aspects of physics, radiobiology, and production of alpha emitters, as well as highlight the main problems they present, with possible new approaches to mitigate those problems. Special emphasis is placed on the strategies proposed for managing recoil energy. We will also provide an account of the recent studies in vitro and in vivo preclinical investigations of α-particle therapy delivered by various nanosystems from different materials, including inorganic nanoparticles, liposomes, and polymersomes, and some carbon-based systems are also summarized.

Preparation and Dosimetry Assessment of 166Dy2O3/166Ho2O3-iPSMA Nanoparticles for Targeted Hepatocarcinoma Radiotherapy.

This research aimed to prepare 166Dy2O3-iPSMA/166Ho2O3-iPSMA nanoparticles (166Dy2O3/166Ho2O3-iPSMA NPs) and assess the radiation absorbed dose produced by the nanosystem to hepatic cancer cells by using experimental in vitro and in vivo biokinetic data. Dy2O3NPs were synthesized and functionalized with the prostate-specific membrane antigen inhibitor peptide (iPSMA). Fourier transform infrared (FTIR) spectroscopy, transmission electron microscope (TEM), dynamic light scattering (DSL) and zeta potential analyses indicated the formation of Dy2O3-iPSMA NPs (46.11 ± 13.24 nm). After neutron activation, a stable 166Dy2O3/166Ho2O3- iPSMA nanosystem was obtained, which showed adequate affinity to the PSMA receptor in HepG2 cancer cells (Kd = 9.87 ± 2.27 nM). in vitro studies indicated high 166Dy2O3/166Ho2O3-iPSMA internalization in cancer cells, with high radiation doses to cell nuclei (107 Gy) and cytotoxic effects, resulting in a significant reduction in HepG2 cell viability (decreasing to 2.12 ± 0.31%). After intratumoral administration in mice, the nanosystem biokinetic profile indicated significant retention into the tumoral mass, producing ablative radiation doses (>70 Gy).

Development of 177Lu-DN(C19)-CXCR4 Ligand Nanosystem for Combinatorial Therapy in Pancreatic Cancer.

Pancreatic cancer is highly lethal and has a poor prognosis. The most common alteration during the formation of pancreatic tumors is the activation of KRAS (Kirsten rat sarcoma 2 viral oncogene homolog) oncogene. As a new therapeutic strategy, the C19 molecule ((2S)-N-(2,5-dichlorophenyl)-2-[(3,4-dimethoxyphenyl)-methylamine]propanamide) blocks the KRAS-membrane association in cancer cells. In addition, the chemokine receptor CXCR4 is overexpressed in pancreatic cancer. In this research, a new dendrimer-based nanoradiopharmaceutical (177Lu-DN(C19)-CXCR4L) encapsulating C19 and functionalized to target CXCR4 receptors is proposed as both, a targeted radiotherapy system (lutetium-177) and an oncotherapeutic approach by the stabilization of KRAS4b-PDESδ complex to produce dual-specific therapy in pancreatic cancer. 177The Lu-DN(C19)-CXCR4L was synthesized and characterized, C19 was encapsulated with 81% efficiency, the final nanosystem rendered a particle size of 67 nm and the specific uptake in pancreatic cell lines was demonstrated. The major cytotoxic effect was observed in the KRAS-dependent and radioresistant cell line Mia PaCa-2, which expresses a high density of CXCR4 receptors. The radiation dose of 3 Gy/Bq decreased viability to 7%, and this effect was attributed to the presence of C19. A synergistic effect (radio and chemotherapy) capable of reducing viability in pancreatic cancer cells through apoptotic mechanisms was demonstrated. Thus, 177Lu-DN(C19)-CXCR4L nanoradiopharmaceutical is efficacious in pancreatic cancer cell lines overexpressing the CXCR4 receptor.

Recent advances in ultrasound-triggered drug delivery through lipid-based nanomaterials.

The high prescribed dose of anticancer drugs and their resulting adverse effects on healthy tissue are significant drawbacks to conventional chemotherapy (CTP). Ideally, drugs should have the lowest possible degree of interaction with healthy cells, which would diminish any adverse effects. Therefore, an ideal scenario to bring about improvements in CTP is the use of technological strategies to ensure the efficient, specific, and selective transport and/or release of drugs to the target site. One practical and feasible solution to promote the efficiency of conventional CTP is the use of ultrasound (US). In this review, we highlight the potential role of US in combination with lipid-based carriers to achieve a targeted CTP strategy in engineered smart drug delivery systems.

In vitro irradiation of doxorubicin with 18F-FDG Cerenkov radiation and its potential application as a theragnostic system.

Doxorubicin (DOX), an effective chemotherapeutic agent, has a wide excitation band centred at 480 nm. Cerenkov radiation (CR) is considered an internal light source in photodynamic therapy (PDT). DOX could be photoactivated by CR and thus, enhancing its cytotoxicity. In this work, 18F-FDG was used to evaluate the effect of Cerenkov radiation on DOX, in comparison to irradiation with a 450-nm laser beam, in terms of ROS production. The production of 1O2 and O2⁎- reactive species during DOX irradiation was detected indirectly by ABMA and DCPIP bleaching, respectively. The cytotoxic effect of the DOX / 18F-FDG CR system was evaluated in the T47D breast cancer cell line. The irradiation of DOX produced 1O2 and O2⁎- species using both 18F-FDG CR and a 450-nm laser beam. The majority reactive species produced in both cases was 1O2; a favourable result, given the greater cytotoxicity of this species. The viability of T47D cells in presence of DOX (5 nM), 18F-FDG (37.5 µCi) and DOX (5 nM)/18F-FDG (37.5 µCi) was (86 ± 9)%, (84 ± 8)% and (64 ± 5)%, respectively; these results suggest a synergistic cytotoxic effect derived from the cytotoxic activity of DOX and its photoactivation by 18F-FDG CR. It is worth noting that the system could be optimized in terms of DOX concentration and 18F-FDG activity for better results. Due to the fact that 18F-FDG is widely used in nuclear imaging, the DOX/18F-FDG system also possesses theragnostic characteristics. Thus, in this work, it is demonstrated that DOX can be used in a dual therapy system based on chemotherapy-PDT when 18F-FDG CR is used as a DOX excitation source.

Synthesis and Evaluation of 177Lu-DOTA-DN(PTX)-BN for Selective and Concomitant Radio and Drug-Therapeutic Effect on Breast Cancer Cells.

The peptide-receptor radionuclide therapy (PRRT) is a successful approach for selectively delivering radiation within tumor sites through specific recognition of radiolabeled peptides by overexpressed receptors on cancer cell surfaces. The efficacy of PRRT could be improved by using polymeric radio- and drug- therapy nanoparticles for a concomitant therapeutic effect on malignant cells. This research aimed to prepare and evaluate, a novel drug and radiation delivery nanosystem based on the 177Lu-labeled polyamidoamine (PAMAM) dendrimer (DN) loaded with paclitaxel (PTX) and functionalized on the surface with the Lys1Lys3(DOTA)-bombesin (BN) peptide for specific targeting to gastrin-releasing peptide receptors (GRPr) overexpressed on breast cancer cells. DN was first conjugated covalently to BN and DOTA (chemical moiety for lutetium-177 complexing) and subsequently loaded with PTX. The characterization by microscopic and spectroscopic techniques, in-vitro drug delivery tests as well as in in-vitro and in-vivo cellular uptake of 177Lu-DOTA-DN(PTX)-BN by T47D breast cancer cells (GRPr-positive), indicated the formation of an improved delivery nanosystem with target-specific recognition by GRPr. Results of the 177Lu-DOTA-DN(PTX)-BN effect on T47D cell viability (1.3%, compared with 10.9% of 177Lu-DOTA-DN-BN and 14.0% of DOTA-DN-(PTX)-BN) demonstrated the concomitant radiotherapeutic and chemotherapeutic properties of the polymeric nanosystem as a potential agent for the treatment of GRPr-positive tumors.

177Lu-Bombesin-PLGA (paclitaxel): A targeted controlled-release nanomedicine for bimodal therapy of breast cancer.

The gastrin-releasing peptide receptor (GRPr) is overexpressed in >75% of breast cancers. 177Lu-Bombesin (177Lu-BN) has demonstrated the ability to target GRPr and facilitate efficient delivery of therapeutic radiation doses to malignant cells. Poly(d,l­lactide­co­glycolide) acid (PLGA) nanoparticles can work as smart drug controlled-release systems activated through pH changes. Considering that paclitaxel (PTX) is a first-line drug for cancer treatment, this work aimed to synthesize and chemically characterize a novel polymeric PTX-loaded nanosystem with grafted 177Lu-BN and to evaluate its performance as a targeted controlled-release nanomedicine for concomitant radiotherapy and chemotherapy of breast cancer. PLGA(PTX) nanoparticles were synthesized using the single emulsification-solvent evaporation method with PVA as a stabilizer in the presence of PTX. Thereafter, the activation of PLGA carboxylic groups for BN attachment through the Lys1-amine group was performed. Results of the chemical characterization by FT-IR, DLS, HPLC and SEM/TEM demonstrated the successful synthesis of BN-PLGA(PTX) with a hydrodynamic diameter of 163.54 ±â€¯33.25 nm. The entrapment efficiency of paclitaxel was 92.8 ±â€¯3.6%. The nanosystem showed an adequate controlled release of the anticancer drug, which increased significantly due to the pH change from neutral (pH = 7.4) to acidic conditions (pH = 5.3). After labeling with 177Lu and purification by ultrafiltration, 177Lu-BN-PLGA(PTX) was obtained with a radiochemical purity of 99 ±â€¯1%. In vitro and in vivo studies using MDA-MB-231 breast cancer cells (GRPr-positive) demonstrated a 177Lu-BN-PLGA(PTX) specific uptake and a significantly higher cytotoxic effect for the radiolabeled nanosystem than the unlabeled BN-PLGA(PTX) nanoparticles. Using a pulmonary micrometastasis MDA-MB-231 model, the added value of 177Lu-BN-PLGA(PTX) for tumor imaging was confirmed. The 177Lu-BN-PLGA(PTX) nanomedicine is suitable as a targeted paclitaxel delivery system with concomitant radiotherapeutic effect for the treatment of GRPr-positive breast cancer.

Preparation and in vitro evaluation of radiolabeled HA-PLGA nanoparticles as novel MTX delivery system for local treatment of rheumatoid arthritis.

Radiosynovectomy is a technique used to decrease inflammation of the synovial tissue by intraarticular injection of a ß-emitting radionuclide, such as 177Lu, which is suitable for radiotherapy due to its decay characteristics. Drug-encapsulating nanoparticles based on poly lactic­co­glycolic acid (PLGA) polymer are a suitable option to treat several arthritic diseases, used as anti-inflammatory drugs transporters of such as methotrexate (MTX), which has been widely used in the arthritis treatment (RA), and hyaluronic acid (HA), which specifically binds the CD44 and hyaluronan receptors overexpressed on the inflamed synovial tissue cells. The 1,4,7,10­Tetraazacyclododecane­1,4,7,10­tetraacetic acid (DOTA) was used as complexing agent of Lutetium-177 for radiotherapy porpoises. The aim of this research was to synthesize 177Lu-DOTA-HA-PLGA(MTX) as a novel, smart drug delivery system with target-specific recognition, potentially useful in radiosynovectomy for local treatment of rheumatoid arthritis. The polymeric nanoparticle system was prepared and chemically characterized. The MTX encapsulation and radiolabelling were performed with suitable characteristics for its in vitro evaluation. The HA-PLGA(MTX) nanoparticle mean diameter was 167.6 nm ±â€¯57.4 with a monomodal and narrow distribution. Spectroscopic techniques demonstrated the effective conjugation of HA and chelating agent DOTA to the polymeric nanosystem. The MTX encapsulation was 95.2% and the loading efficiency was 6%. The radiochemical purity was 96 ±â€¯2%, determined by ITLC. Conclusion: 177Lu-DOTA-HA-PLGA(MTX) was prepared as a biocompatible polymeric PLGA nanoparticle conjugated to HA for specific targeting. The therapeutic nanosystem is based on bi-modal mechanisms using MTX as a disease-modifying antirheumatic drug (DMARD) and 177Lu as a radiotherapeutic component. The 177Lu-DOTA-HA-PLGA(MTX) nanoparticles showed properties suitable for radiosynovectomy and further specific targeted anti-rheumatic therapy.

Synthesis and preclinical evaluation of the 177Lu-DOTA-PSMA(inhibitor)-Lys3-bombesin heterodimer designed as a radiotheranostic probe for prostate cancer.

BACKROUND: Human tumors show intrinsic heterogeneity and changes in phenotype during disease progression, which implies different expression levels of cell surface receptors. The research on new heterodimeric lutetium-177 (Lu)-radiopharmaceuticals interacting with two different targets on tumor cells is a strategy for improvement of radiotheranostic performance. This study aimed to synthesize and characterize the Lu-DOTA-PSMA(inhibitor)-Lys-bombesin (Lu-DOTA-iPSMA-Lys-BN) heterodimer and to evaluate its potential to target prostate-specific membrane antigen (PSMA) and gastrin-releasing peptide receptor (GRPr) overexpressed in prostate cancer. METHODS: The heterodimeric conjugate was synthesized and characterized by infrarred, mass, and H-NMR spectroscopies. The ligand was labeled with Lu and the radiochemical purity was assessed by radio-high-performance liquid chromatography. PSMA/GRPr affinity and the heterobivalent effect on cell viability were evaluated in LNCaP and PC3 prostate cancer cell lines. The biodistribution profile (3 and 96 h) was assessed in athymic mice with induced prostate tumors. Using pulmonary LNCaP (PSMA-positive) and PC3 (GRPr-negative) micrometastasis models, the influence of heterobivalency and affinity on tumor uptake was quantified (micro-SPECT/CT). RESULTS: Lu-iPSMA-BN (radiochemical purity>98%) showed specific recognition for PSMA and GRPr (IC50=5.62 and 3.49 nmol/l, respectively) with a significant decrease in cell viability (10.15% of cell viability in LNCaP and 40.10% in PC3 at 48 h), as well as high LNCaP and PC3 tumor uptake (5.21 and 3.21% ID/g at 96 h, respectively). Micro-SPECT/CT imaging showed the heterodimer ability to target the tumors (SUVmax of 1.93±0.30 and 1.76±0.10 in LNCaP and PC3, respectively), possibly influenced by the heterobivalent effect. Lu-DOTA-iPSMA-Lys-BN showed suitable affinity for PSMA and GRPr. CONCLUSION: The results warrant further preclinical studies to establish the Lu-radiotracer theranostic efficacy.