We have developed herein an engineered polymer-based nanoplatform showing the convergence of two-photon fluorescence imaging and bimodal phototherapeutic activity in a single nanostructure. It was achieved through the appropriate choice of three different components: a β-cyclodextrin-based polymer acting as a suitable carrier, a zinc phthalocyanine emitting red fluorescence simultaneously as being a singlet oxygen ((1)O2) photosensitizer, and a tailored nitroaniline derivative, functioning as a nitric oxide (NO) photodonor. The self-assembly of these components results in photoactivable nanoparticles, approximately 35 nm in diameter, coencapsulating a multifunctional cargo, which can be delivered to carcinoma cells. The combination of steady-state and time-resolved spectroscopic and photochemical techniques shows that the two photoresponsive guests do not interfere with each other while being enclosed in their supramolecular container and can thus be operated in parallel under control of light stimuli. Specifically, two-photon fluorescence microscopy allows mapping of the nanoassembly, here applied to epidermal cancer cells. By detecting the red emission from the phthalocyanine fluorophore it was also possible to investigate the tissue distribution after topical delivery onto human skin ex vivo. Irradiation of the nanoassembly with visible light triggers the simultaneous delivery of cytotoxic (1)O2 and NO, resulting in an amplified cell photomortality due to a combinatory effect of the two cytotoxic agents. The potential of dual therapeutic photodynamic action and two-photon fluorescence imaging capability in a single nanostructure make this system an appealing candidate for further studies in biomedical research.

A water soluble b-CD-branched polymer covalently binds a fluorescein moiety for imaging and a NO photodonor for therapy that can be operated in parallel upon visible light excitation.

We have developed a multifunctional biocompatible nanoconstruct based on polymeric nanoparticles encapsulating a molecular conjugate, able to photorelease nitric oxide (NO) with a fluorescent reporting function. We demonstrate that two-photon excitation (TPE) using biofriendly NIR 700 nm laser light can be applied for monitoring as well as triggering the release of NO, wherein the uncaging of a strongly fluorescent co-product acts in turn as a TPE fluorescent reporter for the simultaneous NO release from the nanoassembly. This supramolecular nanodevice internalizes in skin carcinoma cells, induces significant cell death upon light excitation and preserves its TPE properties, allowing the nearly instantaneous quantification of the NO photoreleased in cancer cells by two-photon NIR fluorescence microscopy.

A supramolecular nanoassembly, of about 30 nm in diameter, that consists of a green‐fluorescent, β‐cyclodextrin‐based, branched polymer co‐encapsulating a red‐emitting singlet oxygen (1O2) photosensitizer and a nitric oxide (NO) photoreleaser, which comprises a blue fluorescent reporter, is here reported. The system exhibits “five‐in‐one” photofunctionalities. All components can be simultaneously excited in the phototherapeutic window with two‐photons by using near‐infrared light at 740 nm and despite their close proximity, behave as independent units. This allows for their in vitro visualization in carcinoma cancer cells, due to their distinct green, red, and blue fluorescence, and for the production of both cytotoxic 1O2 and biofunctional NO.

Gold nanoparticles can be visualized in far-field multiphoton laser-scanning microscopy (MPM) based on the phenomena of multiphoton induced luminescence (MIL). This is of interest for biomedical applications, e.g., for cancer diagnostics, as MPM allows for working in the near-infrared (NIR) optical window of tissue. It is well known that the aggregation of particles causes a redshift of the plasmon resonance, but its implications for MIL applying far-field MPM should be further exploited. Here, we explore MIL from 10 nm gold nanospheres that are chemically deposited on glass substrates in controlled coverage gradients using MPM operating in NIR range. The substrates enable studies of MIL as a function of inter-particle distance and clustering. It was shown that MIL was only detected from areas on the substrates where the particle spacing was less than one particle diameter, or where the particles have aggregated. The results are interpreted in the context that the underlying physical phenomenon of MIL is a sequential two-photon absorption process, where the first event is driven by the plasmon resonance. It is evident that gold nanospheres in this size range have to be closely spaced or clustered to exhibit detectable MIL using far-field MPM operating in the NIR region.