18F-Radiolabeled porous silicon particles for drug delivery : Tracer development and evaluation in rats

Poor biopharmaceutical properties such as low solubility and low permeability in the gastrointestinal (GI) tract plight many existing drugs and new chemical entities, presenting an impediment for efficient drug therapy. Incorporation of the drug to a delivery system based on a nanostructured material is increasingly investigated as a strategy to overcome these limitations and to achieve controlled and targeted delivery. Porous silicon (PSi) is a promising material for carrier-mediated drug delivery because of its biocompatibility, high chemical stability, and facile elimination from the body. Moreover, the physicochemical properties of PSi can be tailored by variation of the fabrication parameters and surface modifications to suit diverse payloads. Positron emission tomography (PET), a sensitive and quantitative method of molecular imaging, is a potent tool for drug delivery system development. Already at the preclinical stage PET can be employed for the investigation of drug delivery carrier biodistribution in vivo, thereby facilitating the selection of the most promising material candidates for further development and future drug delivery studies. In this dissertation, a direct nucleophilic radiolabeling method with a short-lived positron emitter fluorine-18 (18F) was developed for three different surface-modified PSi materials: thermally hydrocarbonized PSi (THCPSi), thermally carbonized PSi (TCPSi), and thermally oxidized PSi (TOPSi). Out of the investigated materials, nanosized [18F]THCPSi emerged as the one with the highest potential for imaging and drug delivery in terms radiolabeling yield, label stability, and biocompatibility in cell models in vitro, and was therefore forwarded to biodistribution studies in rats. After oral administration, [18F]THCPSi nanoparticles were shown to pass intact through the GI tract in 4 to 6 hours. Modification of [18F]THCPSi with a self-assembled layer of a fungal hydrophobin (HFBII) changed the hydrophilicity of the material bringing about bioadhesive properties that promoted gastric retention of the protein-coated nanoparticles. Intravenous delivery of [18F]THCPSi nanoparticles resulted in their rapid accumulation to the liver and spleen alluding to rapid immune recognition and removal of the particles from the bloodstream by macrophages of the mononuclear phagocyte system (MPS). HFBII-coating of the nanoparticles altered the adsorption of plasma proteins to the particle surface, which translated also to a change in the biodistribution pattern in vivo. In conclusion, the present work establishes 18F-radiolabeled particle tracers as useful means for the evaluation of new PSi-based drug delivery systems with PET.Lääkeaineen biofarmaseuttisesti epäedulliset ominaisuudet, kuten niukkaliukoisuus ja huono permeabiliteetti ruoansulatuskanavassa voivat muodostua esteeksi tehokkaalle lääkehoidolle. Eräs uusi strategia näiden ongelmien ratkaisuun on lääkeaineen liittäminen nanorakenteiseen lääkeainekuljettimeen, jonka avulla sitä voidaan annostella säädellysti sekä kohdennetusti. Huokoinen pii (PSi) on lupaava materiaali lääkeainekuljettimien kehitykseen, sillä sen on osoitettu olevan hyvin bioyhteensopivaa, biohajoavaa sekä kemiallisesti stabiilia. Tämän lisäksi huokoisen piin fysikaalis-kemiallisia ominaisuuksia voidaan räätälöidä sen valmistusparametrejä muokkaamalla sekä erilaisin pintamodifikaatioin soveltumaan useiden erilaisten aineiden kuljetukseen. Positroniemissiotomografia (PET) on herkkä ja kvantitatiivinen molekyylikuvantamisen menetelmä, joka soveltuu hyvin lääkeainekuljettimien kehitystyön tueksi. Jo kehityksen prekliinisessä vaiheessa positroniemissiotomografiaa voidaan käyttää lääkeainekuljettimen biojakauman selvittämiseen in vivo, edesauttaen lupaavimpien materiaalikandidaattien valintaa jatkokehitykseen ja tuleviin lääkeaineiden annostelukokeisiin. Tässä väitöskirjassa kehitettiin suora nukleofiilinen radioleimausmenetelmä lyhytikäisellä positronisäteilijällä, fluori-18:lla (18F) kolmelle erilaiselle pintamuokatulle PSi-materiaalille (THCPSi, TCPSi ja TOPSi). Tutkituista materiaaleista nanokokoinen termisesti hydrokarbidoitu [18F]THCPSi osoittautui soveltuvimmaksi kuvantamiseen ja lääkeainekuljetukseen perustuen sen radioleimauksen saantoon, leiman stabiilisuuteen sekä bioyhteensopivuuteen solumalleissa. Oraalisen annon jälkeen [18F]THCPSi -nanopartikkelit kulkeutuivat muuttumattomina läpi rotan ruoansulatuskanavan 4 - 6 tunnissa. [18F]THCPSi:n muokkaus itsejärjestäytyvällä kerroksella sienestä peräisin olevaa hydrofobiinia (HFBII) muutti materiaalin hydrofiilisyyttä ja teki siitä bioadhesiivisen. Nämä muutokset johtivat proteiinilla muokattujen nanopartikkelienpidättymiseen mahaan. Suonensisäisen annon jälkeen [18F]THCPSi -nanopartikkelit kertyivät maksaan ja pernaan, mikä viittaa niiden tunnistamiseen immuunijärjestelmässä ja siitä seuraavaan nopeaan poistoon verenkierrosta näissä elimissä olevien makrofagien toimesta. Päällystäminen hydrofobiinilla johti muutoksiin veriplasman proteiinien adsorptiossa nanopartikkelien pinnalle. Tämän seurauksena myös biodistribuutiossa havaittiin muutos in vivo. Johtopäätöksenä todettakoon, että tässä työssä 18F-radioleimatut partikkelimerkkiaineet on osoitettu hyödyllisiksi työkaluiksi uusien PSi-pohjaisten lääkeainekuljettimien arviointiin positroniemissiotomografian avulla

Radiolabeling of Theranostic Nanosystems

In the recent years, progress in nanotechnology has significantly contributed to the development of novel pharmaceutical formulations to overcome the drawbacks of conventional treatments and improve the therapeutic outcome in many diseases, especially cancer. Nanoparticle vectors have demonstrated the potential to concomitantly deliver diagnostic and therapeutic payloads to diseased tissue. Due to their special physical and chemical properties, the characteristics and function of nanoparticles are tunable based on biological molecular targets and specific desired features (e.g., surface chemistry and diagnostic radioisotope labeling). Within the past decade, several theranostic nanoparticles have been developed as a multifunctional nanosystems which combine the diagnostic and therapeutic functionalities into a single drug delivery platform. Theranostic nanosystems can provide useful information on a real-time systemic distribution of the developed nanosystem and simultaneously transport the therapeutic payload. In general, the diagnostic functionality of theranostic nanoparticles can be achieved through labeling gamma-emitted radioactive isotopes on the surface of nanoparticles which facilitates noninvasive detection using nuclear molecular imaging techniques, such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT), meanwhile, the therapeutic effect arises from the potent drug released from the nanoparticle. Moreover, some radioisotopes can concurrently emit both gamma radiation and high-energy particles (e.g., alpha, beta, and Auger electrons), prompting the use either alone for radiotheranostics or synergistically with chemotherapy. This chapter provides an overview of the fundamentals of radiochemistry and relevant radiolabeling strategies for theranostic nanosystem development as well as the methods for the preclinical evaluation of radiolabeled nanoparticles. Furthermore, preclinical case studies of recently developed theranostic nanosystems will be highlighted.Peer reviewe

Evaluation of Organo [18F]Fluorosilicon Tetrazine as a Prosthetic Group for the Synthesis of PET Radiotracers

Fluorine-18 is the most widely used positron emission tomography (PET) radionuclide currently in clinical application, due to its optimal nuclear properties. The synthesis of 18F-labeled radiotracers often requires harsh reaction conditions, limiting the use of sensitive bio- and macromolecules as precursors for direct radiolabeling with fluorine-18. We aimed to develop a milder and efficient in vitro and in vivo labeling method for trans-cyclooctene (TCO) functionalized proteins, through the bioorthogonal inverse-electron demand Diels-Alder (IEDDA) reaction with fluorine-18 radiolabeled tetrazine ([18F]SiFA-Tz). Here, we used TCO-modified bovine serum albumin (BSA) as the model protein, and isotopic exchange (IE) (19F/18F) chemistry as the labeling strategy. The radiolabeling of albumin-TCO with [18F]SiFA-Tz ([18F]6), providing [18F]fluoroalbumin ([18F]10) in high radiochemical yield (99.1 ± 0.2%, n = 3) and a molar activity (MA) of 1.1 GBq/µmol, confirmed the applicability of [18F]6 as a quick in vitro fluorination reagent for the TCO functionalized proteins. While the biological evaluation of [18F]6 demonstrated defluorination in vivo, limiting the utility for pretargeted applications, the in vivo stability of the radiotracer was dramatically improved when [18F]6 was used for the radiolabeling of albumin-TCO ([18F]10) in vitro, prior to administration. Due to the detected defluorination in vivo, structural optimization of the prosthetic group for improved stability is needed before further biological studies and application of pretargeted PET imaging

Efficient cartridge purification for producing high molar activity [18F]fluoro-glycoconjugates via oxime formation

Introduction 18F-fluoroglycosylation via oxime formation is a chemoselective and mild radiolabeling method for sensitive molecules. Glycosylation can also improve the bioavailability, in vivo kinetics, and stability of the compound in blood, as well as accelerate clearance of biomolecules. A typical synthesis procedure for 18F-fluoroglycosylation with [18F]FDG (2-deoxy-2-[18F]fluoro-d-glucose) and [18F]FDR (5-deoxy-5-[18F]fluoro-d-ribose) involves two HPLC (high performance liquid chromatography) purifications: one after 18F-fluorination of the carbohydrate to remove its labeling precursor, and a second one after the oxime formation step to remove the aminooxy precursor. The two HPLC purifications can be time consuming and complicate the adaptation of the synthetic strategy in nuclear medicine applications and automated synthesis. We have developed a procedure in which SPE (solid phase extraction) and resin purification methods replace both of the needed HPLC purification steps. Methods We used [18F]FDR and [18F]FDG as prosthetic groups to radiolabel two aminooxy-modified model molecules, a tetrazine and a PSMA (prostate specific membrane antigen) inhibitor. After fluorination, the excess carbohydrate precursor was removed by derivatizing it with 4,4′-dimethoxytrityl chloride (DMT-Cl). The DMT moiety increases the hydrophobicity of the unreacted precursor making the separation from the fluorinated precursor possible with simple C18 Sep-Pak cartridge. For removal of the aminooxy precursor, we used a commercially available aldehyde resin (AminoLink, Thermo Fisher Scientific). C18 Sep-Pak SPE cartridge was used to separate [18F]FDR and [18F]FDG from the 18F-fluoroglycoconjugate end product. Results [18F]FDR and [18F]FDG were efficiently purified from their precursors, free fluorine-18, and other impurities. The aldehyde resin quantitatively removed the unreacted aminooxy precursors after the oxime formation. The fluorine-18 labeled oxime end products were obtained with high radiochemical purity (>99%) and molar activity (>600 GBq μmol−1). Conclusions We have developed an efficient cartridge purification method for producing high molar activity 18F-glycoconjugates synthesized via oxime formation.Peer reviewe

Evaluation of Organo [18F]Fluorosilicon Tetrazine as a Prosthetic Group for the Synthesis of PET Radiotracers

Fluorine-18 is the most widely used positron emission tomography (PET) radionuclide currently in clinical application, due to its optimal nuclear properties. The synthesis of 18F-labeled radiotracers often requires harsh reaction conditions, limiting the use of sensitive bio- and macromolecules as precursors for direct radiolabeling with fluorine-18. We aimed to develop a milder and efficient in vitro and in vivo labeling method for trans-cyclooctene (TCO) functionalized proteins, through the bioorthogonal inverse-electron demand Diels-Alder (IEDDA) reaction with fluorine-18 radiolabeled tetrazine ([18F]SiFA-Tz). Here, we used TCO-modified bovine serum albumin (BSA) as the model protein, and isotopic exchange (IE) (19F/18F) chemistry as the labeling strategy. The radiolabeling of albumin-TCO with [18F]SiFA-Tz ([18F]6), providing [18F]fluoroalbumin ([18F]10) in high radiochemical yield (99.1 ± 0.2%, n = 3) and a molar activity (MA) of 1.1 GBq/µmol, confirmed the applicability of [18F]6 as a quick in vitro fluorination reagent for the TCO functionalized proteins. While the biological evaluation of [18F]6 demonstrated defluorination in vivo, limiting the utility for pretargeted applications, the in vivo stability of the radiotracer was dramatically improved when [18F]6 was used for the radiolabeling of albumin-TCO ([18F]10) in vitro, prior to administration. Due to the detected defluorination in vivo, structural optimization of the prosthetic group for improved stability is needed before further biological studies and application of pretargeted PET imaging

Site-Specific 111In-Radiolabeling of Dual-PEGylated Porous Silicon Nanoparticles and Their In Vivo Evaluation in Murine 4T1 Breast Cancer Model

Polyethylene glycol (PEG) has been successfully used for improving circulation time of several nanomaterials but prolonging the circulation of porous silicon nanoparticles (PSi NPs) has remained challenging. Here, we report a site specific radiolabeling of dual-PEGylated thermally oxidized porous silicon (DPEG-TOPSi) NPs and investigation of influence of the PEGylation on blood circulation time of TOPSi NPs. Trans-cyclooctene conjugated DPEG-TOPSi NPs were radiolabeled through a click reaction with [111In]In-DOTA-PEG4-tetrazine (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and the particle behavior was evaluated in vivo in Balb/c mice bearing 4T1 murine breast cancer allografts. The dual-PEGylation significantly prolonged circulation of [111In]In-DPEG-TOPSi particles when compared to non-PEGylated control particles, yielding 10.8 ± 1.7% of the injected activity/g in blood at 15 min for [111In]In-DPEG-TOPSi NPs. The improved circulation time will be beneficial for the accumulation of targeted DPEG-TOPSi to tumors

Site-Specific 111In-Radiolabeling of Dual-PEGylated Porous Silicon Nanoparticles and Their In Vivo Evaluation in Murine 4T1 Breast Cancer Model

Polyethylene glycol (PEG) has been successfully used for improving circulation time of several nanomaterials but prolonging the circulation of porous silicon nanoparticles (PSi NPs) has remained challenging. Here, we report a site specific radiolabeling of dual-PEGylated thermally oxidized porous silicon (DPEG-TOPSi) NPs and investigation of influence of the PEGylation on blood circulation time of TOPSi NPs. Trans-cyclooctene conjugated DPEG-TOPSi NPs were radiolabeled through a click reaction with [111In]In-DOTA-PEG4-tetrazine (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and the particle behavior was evaluated in vivo in Balb/c mice bearing 4T1 murine breast cancer allografts. The dual-PEGylation significantly prolonged circulation of [111In]In-DPEG-TOPSi particles when compared to non-PEGylated control particles, yielding 10.8 ± 1.7% of the injected activity/g in blood at 15 min for [111In]In-DPEG-TOPSi NPs. The improved circulation time will be beneficial for the accumulation of targeted DPEG-TOPSi to tumors

Multimodality labeling strategies for the investigation of nanocrystalline cellulose biodistribution in a mouse model of breast cancer

Methods We have developed a nuclear and fluorescence labeling strategy for nanocrystalline cellulose (CNC), an emerging biomaterial with versatile chemistry and facile preparation from renewable sources. We modified CNC through 1,1′-carbonyldiimidazole (CDI) activation with radiometal chelators desferrioxamine B and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), allowing for the labeling with zirconium-89 (t½ = 78.41 h) and copper-64 (t½ = 12.70 h), respectively, for non-invasive positron emission tomography (PET) imaging. The far-red fluorescent dye Cy5 was added for ex vivo optical imaging, microscopy and flow cytometry. The multimodal CNC were evaluated in the syngeneic orthotopic 4T1 tumor model of human stage IV breast cancer. Results Modified CNC exhibited low cytotoxicity in RAW 264.7 macrophages over 96 h, and high radiolabel stability in vitro. After systemic administration, radiolabeled CNC were rapidly sequestered to the organs of the reticulo-endothelial system (RES), indicating immune recognition and no passive tumor targeting by the enhanced permeability and retention (EPR) effect. Modification with NOTA was a more favorable strategy in terms of radiolabeling yield, specific radioactivity, and both the radiolabel and dispersion stability in physiological conditions. Flow cytometry analysis of Cy5-positive immune cells from the spleen and tumor corroborated the uptake of CNC to phagocytic cells. Conclusions Future studies on the in vivo behavior of CNC should be concentrated on improving the nanomaterial stability and circulation half-life under physiological conditions and optimizing further the labeling yields for the multimodality imaging strategy presented. Advances in knowledge Our studies constitute one of the first accounts of a multimodality nuclear and fluorescent probe for the evaluation of CNC biodistribution in vivo and outline the pitfalls in radiometal labeling strategies for future evaluation of targeted CNC-based drug delivery systems. Implications for patient care Quantitative and sensitive molecular imaging methods provide information on the structure–activity relationships of the nanomaterial and guide the translation from in vitro models to clinically relevant animal models.Peer reviewe

Evaluation of organo [18F]fluorosilicon tetrazine as a prosthetic group for the synthesis of PET radiotracers

Fluorine-18 is the most widely used positron emission tomography (PET) radionuclide currently in clinical application, due to its optimal nuclear properties. The synthesis of 18F-labeled radiotracers often requires harsh reaction conditions, limiting the use of sensitive bio- and macromolecules as precursors for direct radiolabeling with fluorine-18. We aimed to develop a milder and efficient in vitro and in vivo labeling method for trans-cyclooctene (TCO) functionalized proteins, through the bioorthogonal inverse-electron demand Diels-Alder (IEDDA) reaction with fluorine-18 radiolabeled tetrazine ([18F]SiFA-Tz). Here, we used TCO-modified bovine serum albumin (BSA) as the model protein, and isotopic exchange (IE) (19F/18F) chemistry as the labeling strategy. The radiolabeling of albumin-TCO with [18F]SiFA-Tz ([18F]6), providing [18F]fluoroalbumin ([18F]10) in high radiochemical yield (99.1 ± 0.2%, n = 3) and a molar activity (MA) of 1.1 GBq/µmol, confirmed the applicability of [18F]6 as a quick in vitro fluorination reagent for the TCO functionalized proteins. While the biological evaluation of [18F]6 demonstrated defluorination in vivo, limiting the utility for pretargeted applications, the in vivo stability of the radiotracer was dramatically improved when [18F]6 was used for the radiolabeling of albumin-TCO ([18F]10) in vitro, prior to administration. Due to the detected defluorination in vivo, structural optimization of the prosthetic group for improved stability is needed before further biological studies and application of pretargeted PET imaging.</p

Development of 68Ga-labeled Hepatitis E virus nanoparticles for targeted drug delivery and diagnostics with PET

Peer reviewe