Core-shell polymeric nanoparticles co-loaded with photosensitizer and organic dye for photodynamic therapy guided by fluorescence imaging in near and short-wave infrared spectral regions.

BACKGROUND: Biodistribution of photosensitizer (PS) in photodynamic therapy (PDT) can be assessed by fluorescence imaging that visualizes the accumulation of PS in malignant tissue prior to PDT. At the same time, excitation of the PS during an assessment of its biodistribution results in premature photobleaching and can cause toxicity to healthy tissues. Combination of PS with a separate fluorescent moiety, which can be excited apart from PS activation, provides a possibility for fluorescence imaging (FI) guided delivery of PS to cancer site, followed by PDT. RESULTS: In this work, we report nanoformulations (NFs) of core-shell polymeric nanoparticles (NPs) co-loaded with PS [2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a, HPPH] and near infrared fluorescent organic dyes (NIRFDs) that can be excited in the first or second near-infrared windows of tissue optical transparency (NIR-I, ~ 700-950 nm and NIR-II, ~ 1000-1350 nm), where HPPH does not absorb and emit. After addition to nanoparticle suspensions, PS and NIRFDs are entrapped by the nanoparticle shell of co-polymer of N-isopropylacrylamide and acrylamide [poly(NIPAM-co-AA)], while do not bind with the polystyrene (polySt) core alone. Loading of the NIRFD and PS to the NPs shell precludes aggregation of these hydrophobic molecules in water, preventing fluorescence quenching and reduction of singlet oxygen generation. Moreover, shift of the absorption of NIRFD to longer wavelengths was found to strongly reduce an efficiency of the electronic excitation energy transfer between PS and NIRFD, increasing the efficacy of PDT with PS-NIRFD combination. As a result, use of the NFs of PS and NIR-II NIRFD enables fluorescence imaging guided PDT, as it was shown by confocal microscopy and PDT of the cancer cells in vitro. In vivo studies with subcutaneously tumored mice demonstrated a possibility to image biodistribution of tumor targeted NFs both using HPPH fluorescence with conventional imaging camera sensitive in visible and NIR-I ranges (~ 400-750 nm) and imaging camera for short-wave infrared (SWIR) region (~ 1000-1700 nm), which was recently shown to be beneficial for in vivo optical imaging. CONCLUSIONS: A combination of PS with fluorescence in visible and NIR-I spectral ranges and, NIR-II fluorescent dye allowed us to obtain PS nanoformulation promising for see-and-treat PDT guided with visible-NIR-SWIR fluorescence imaging.

Hyperspectral Multiplexed Biological Imaging of Nanoprobes Emitting in the Short-Wave Infrared Region.

Optical bioimaging with exogenous luminophores emitting in short-wave infrared spectral region (SWIR, ~ 1000-1700 nm) is a rapidly developing field, and the development of multiple SWIR-photoluminescent nanoprobes has recently been reported. In this regard, hyperspectral imaging (HSI), combined with unmixing algorithms, is a promising tool that can allow for efficient multiplexing of the SWIR-emitting nanoagents by their photoluminescence (PL) spectral profiles. The SWIR HSI technique reported here is developed to multiplex two types of nanoprobes: polymeric nanoparticles doped with organic dye (PNPs) and rare-earth doped fluoride nanoparticles (RENPs). Both types of nanoprobes exhibit PL in the same spectral range (~ 900-1200 nm), which hinders spectral separation of PL with optical filters and limits possibilities for their multiplexed imaging in biological tissues. By applying SWIR HSI, we exploited differences in the PL spectral profiles and achieved the spectrally selective and sensitive imaging of the PL signal from every type of nanoparticles. Unmixing of acquired data allowed for multiplexing of the spectrally overlapping nanoprobes by their PL profile. Both quantitative and spatial distribution for every type of nanoparticles were obtained from their mixed suspensions. Finally, the SWIR HSI technique with unmixing protocol was applied to in vivo imaging of mice subcutaneously injected with PNPs and RENPs. The applicability of hyperspectral techniques to multiplex nanoprobes in the in vivo imaging was successfully demonstrated.