Mixed β-γ-Cyclodextrin Branched Polymer with Multiple Photo-Chemotherapeutic Cargos

The achievement of biocompatible platforms for multimodal therapies is one of the major challenges in the burgeoning field of nanomedicine. Here, we report on a mixed β- and γ-cyclodextrin-based branched polymeric material (βγCD-NOPD) covalently integrating a nitric oxide (NO) photodonor (NOPD) within its macromolecular scaffold, and its supramolecular ensemble with a singlet oxygen (1O2) photosensitizer (PS) Zn(II) phthalocyanine (ZnPc) and the chemodrug Lenvatinib (LVB). This polymer is highly water-soluble and generates NO under visible blue light stimuli with an efficiency of more than 1 order of magnitude higher than that of the single NOPD. The PS, which in an aqueous solution is aggregated and non-photoresponsive, can be entangled in the polymeric network as a photoresponsive monomeric species. In addition, the poorly water-soluble LVB can be co-encapsulated within the polymeric host, which increases the drug solubility by more than 30-fold compared to the free drug and more than 2-fold compared with a similar branched polymer containing only βCD units. The supramolecular nanoensemble, ca. 15 nm in diameter, retains well the photochemical properties of both the NOPD and PS, which can operate in parallel under light stimuli of different energies. Irradiation with blue and red light results in the photogeneration of NO and 1O2 associated with red fluorescence emission, without inducing any photodegradation of LVB. This result is not trivial and is due to the absence of significant, mutual interactions between the NOPD, the PS and LVB both in the ground and excited states, despite these components are confined in the same host. The proposed polymeric nanoplatform may represent a potential trimodal nanomedicine for biomedical research studies, since it combines the double photodynamic action of NO and 1O2, two species that do not suffer multidrug resistance, with the therapeutic activity of a conventional chemodrug.


INTRODUCTION
Multimodal therapies to fight cancer diseases exploit different treatment modalities with the goal of improving the therapeutic outcome through synergistic/additive actions while minimizing side effects. 1,2In this regard, a combination of conventional chemotherapeutics with unconventional lightgenerated therapeutics is a very appealing approach that has been receiving growing attention, especially over the last decade. 3,4Light is an ideal tool for the introduction of a cytotoxic species in a bioenvironment with very precise control of site and dosage through the straightforward regulation of the irradiated area and irradiation time. 5,6mong light-stimulated unconventional approaches to fight cancers, photodynamic therapy (PDT) represents the most promising and it also finds application in clinics. 7,8PDT is mainly based on the cytotoxic action of the highly reactive singlet oxygen ( 1 O 2 ). 9 This species is much more oxidant than molecular oxygen and is generated in a catalytic fashion by collisional energy transfer between the excited triplet state of a photosensitizer (PS) and the molecular oxygen. 10n recent years, photodynamic treatments exploiting nitric oxide (NO) as unconventional therapeutics, namely, NO-PDT, have come overwhelmingly into the limelight and hold very promising features in cancer, although still confined to the research area. 11In addition to being a key bioregulator of a broad array of physiological processes, 12 NO can act as an efficient antitumoral agent if produced at a suitable dosage.Micromolar concentrations of this diatomic free radical induce cell toxicity 13 and inhibit the efflux pumps mainly responsible for multidrug resistance (MDR) in cancer cells. 14−19 However, it is also known that doses of NO produced in the nanomolar range can induce tumor proliferation. 20−24 Different from 1 O 2 , NO is not generated through a catalytic mechanism.−24 Therefore, the reservoir of NO is strictly dictated by the initial concentration of NOPDs.
Note that 1 O 2 and NO are multitarget "bullets" that do not suffer MDR phenomena and due to their short lifetimes (few μs and few s, respectively) confine their reactivity to short distances from their generation site (0.02−200 μm), minimizing systemic toxicity drawbacks typical of many chemotherapeutic drugs.Finally, NO photorelease does not depend on the presence of molecular oxygen, making NO-PDT complementary to PDT under hypoxia conditions, which are typical for some tumors.
On these grounds, the achievement of multifunctional supramolecular assemblies able to photogenerate 1 O 2 and NO and simultaneously incorporate conventional chemotherapeutics is a very challenging objective to pursue.In this regard, the significant breakthroughs in nanomedicine offer an unprecedented opportunity to design and fabricate polymeric nanoplatforms able to entrap multiple therapeutic/photo-therapeutic agents in a single nanocarrier. 25,26This permits, in principle, the controlled delivery of several therapeutics in the same bioenvironment, encouraging their action on either a single oncogenic pathway through different mechanisms or across parallel pathways.
Branched polymers 27 are impetuously emerging in view of their unique topological structures and physicochemical properties such as three-dimensional globular structure, small hydrodynamic radius, improved multifunctionality, and excellent encapsulation capabilities and water solubility, as recently described by Vicent and co-workers in a recent review paper. 28−31 CDs are cyclic oligosaccharides based on 6 (αCD), 7 (βCD), or 8 (γCD) glucopyranose units, well known for their complexation, stabilization, and solubilization capabilities of a wide range of guest compounds. 32The most extensively used cross-linking reagent for the achievement of branched CD polymers is epichlorohydrin. 33−31 We have demonstrated their suitability as host scaffolds for multimodal therapies with supramolecular systems combining PDT and NO-PDT 34−37 as well as NO-PDT and chemotherapy. 18,19Recently, an interesting class of CD-based branched polymers containing both β and γCD units in the same macromolecular scaffold and obtained by green synthetic protocols has been reported. 38These mixed polymeric hosts offer a large number of binding sites with differentiated sizes and hydrophobic/hydrophilic features if compared with the analogues containing a single type of CD, permitting an easier allocation of multiple guests in different compartments. 39In Scheme 1. Top: Molecular Structures of the Mixed-Branched Polymer βγCD-NOPD, the PS ZnPc, the Chemodrug LVB, and the Model NOPD-1.Bottom: Schematic View of the Trimodal Supramolecular Assembly the case of photoactivatable systems, this peculiarity assumes particular relevance because, in principle, it better avoids mutual guest interactions in the ground and excited states, which otherwise would modify the response to light in efficiency, nature, or both.
With the aim to move toward trimodal photo-chemotherapeutic platforms, we report herein a mixed βand γCDbased branched polymeric material βγCD-NOPD, which covalently integrates a nitroaniline derivative as NOPD within its macromolecular skeleton (Scheme 1).We demonstrate that this polymer (i) releases NO under blue light with efficiency higher than that of the individual NOPD unit, (ii) encapsulates the Zn(II) phthalocyanine tetrasulfonate ZnPc, a PS for PDT totally aggregated and non-photoresponsive in water, mainly under its photoresponsive monomeric form, (iii) coencapsulates the poorly water-soluble chemotherapeutic Lenvatinib (LVB), increasing its solubility of ∼35-fold compared with the free drug, and (iv) preserves well the individual properties of all photo-chemotherapeutic components despite their confinement within the same host (Scheme 1).To our knowledge, no similar systems are known to date.

βγCD-NOPD and Its Binary Assemblies with LVB
and ZnPc.βγCD-NOPD was synthesized in one step from the copolymerization of a functionalized βCD monomer and native γCD using epichlorohydrin as a cross-linker (Scheme 2) and described in detail in the Supporting Information.The functionalized βCD monomer integrates two units of a 4-nitro-3-(trifluoromethyl)amino-derivative, an NOPD developed in our group, 40 which have been covalently linked to the lower rim of the macrocycle through click-chemistry starting from suitable precursors (see the Supporting Information).Analogously to other sterically hindered ortho-substituted nitroderivatives, 41 this NOPD uncages NO under blue light irradiation through a mechanistic pathway involving a nitroto-nitrite rearrangement, the homolytic rupture of the O−NO bond and the formation of a phenoxy radical (inset of Scheme 2).βγCD-NOPD (M W ca. 70 kD) contains ca.1.8% (w/w) of NOPD and is highly soluble in aqueous media.Figure 1A shows the UV−vis absorption spectra of the polymer and, for the sake of comparison, that of the water-soluble model NOPD-1 (see Scheme 1).The absorption features of βγCD-NOPD are dominated by the large band of the NOPD unit in the blue region with a maximum at 393 nm and extending up to 500 nm.Note that, although the spectral shape profile is basically unchanged, the band of the NOPD moiety in the case of the polymer is shifted to the blue of ca. 4 nm if compared with the model compound.Due to the large charge transfer character of this band, the observed shift agrees with a less polar environment experienced by the NOPD moiety, which results in not complete exposure to the water pool but a partial embedding within more hydrophobic compartments of the polymeric network.
Irradiation of βγCD-NOPD leads to the bleaching of the main absorption band with a photolysis rate independent of the presence of oxygen (Figure 1B and related inset).This photobehavior is in excellent agreement with that shown by the individual NOPD, 40 accounting for the loss of NO upon light irradiation and confirming that the integration of the NOPD unit within the polymeric scaffold does not change the nature of the primary photochemical process.
Photostimulated NO release was confirmed by direct amperometric detection.Figure 1C clearly shows that NO production is strictly dependent on the irradiation conditions as demonstrated by the alternate light/dark cycles.Interestingly, we obtained a value for the NO photorelease quantum yield Φ NO = 0.007 ± 0.001, which is more than 1 order of magnitude higher than observed for the model NOPD-1 under the same irradiation conditions (see traces in Figure 1C).This value is in good agreement with what has been recently observed for NOPDs based on the same chromophore covalently linked to branched polymers based only on βCD units 19,37 or entrapped in micellar hosts. 42,43This enhancement of photoreactivity is the result of the active role of the CD units of the polymer as a reactant, providing H-atoms to the phenoxy radical involved in the NO photorelease mechanism (see Scheme 2).
βγCD-NOPD emerged as a good host to encapsulate the poorly water-soluble LVB, whose solubility in this solvent is ∼4 μM (∼1.7 μg mL −1 ). Figure 2 shows that the drug solubility significantly increases in the presence of the polymer, reaching a value of ca.125 μM (ca.65 μg mL −1 ) (inset Figure 2).Such a value is ca.30-fold higher than that observed in the absence of the polymer and more than 2-fold higher than that observed for the same drug in the presence of the same amount of a branched polymer containing only βCD units. 19e obtained values of ca.97% for the encapsulation efficiency and ca.3.3% for drug loading (see the Supporting Information).
Note that the absorption profile of the encapsulated LVB is basically identical to that observed in the methanol solution (see spectrum h in Figure 2), accounting for the lack of any intrahost aggregation of the chemodrug.Moreover, the absorption spectra of Figure 2 in the whole concentration range explored did not change for several days, indicating good stability of the complex under ambient conditions.
βγCD-NOPD was also a very suitable host to interact with the highly hydrophilic ZnPc (see Scheme 1).This compound is a well-known PS for PDT, being able to generate the cytotoxic 1 O 2 for therapy and to emit red fluorescence useful for imaging under red-light excitation. 44ZnPc is very soluble in aqueous solution, where it shows absorption bands at 335 and 635 nm, respectively (spectrum a in Figure 3).However, the formation of water-soluble aggregates 45 precludes its response to light, resulting in the ineffective population of the triplet state, consequent lack of 1 O 2 photogeneration, and very low fluorescence emission in this solvent. 46,47Addition of βγCD-NOPD to a water solution of ZnPc significantly breaks its selfaggregation, encouraging the entangling of the PS within the polymeric network as a monomer in a satisfactory amount (≥20%).This is confirmed by the decrease of the absorption band of the aggregate form at 635 nm and the concomitant formation of a new absorption band with λ max = 680 nm, typical for the monomeric species 45 (spectrum b in Figure 3).In addition, a significant increase in the typical red emission of ZnPc when compared to that in the absence of polymer was observed (spectra c and d in Figure 3).The fluorescence decay is characterized by a dominant component (≥84%) with the lifetime τ = 3.5 ns (inset Figure 3).Note that the emission spectra observed in the presence of the polymer are red-shifted ca.7 nm when compared with the free ZnPc.This is the result  of environmental effects on the photophysical features of the PS, in accordance with its red-shifted emission observed in nonaqueous media compared to water solution. 48As previously reported, the entrapment of momomeric ZnPc can be encouraged by the three-dimensional (3D) structure of the CD polymer in which the high local concentration of CD nanocavities in the cross-linked network may act cooperatively in the disruption of the aggregate forms. 48In principle, ZnPc is able to interact with the polymer considering that the benzene sulfonate moieties attached to its macrocyclic structure are available to interact with the β and γCD units due to their favorable geometrical matching. 49,50n addition to restoring the fluorescence emission of ZnPc, the branched polymeric host leads to the population of the lowest triplet state of the PS, which is a key transient intermediate involved in the photogeneration of the cytotoxic 1 O 2 through collisional energy transfer with molecular oxygen. 7,9Nanosecond laser flash photolysis is a powerful tool to obtain direct evidence on spectroscopic and kinetic features of the triplet states of the porphyrinoid systems.They show in fact intense absorptions in the visible region and have lifetimes falling in the microsecond time regime. 51In contrast to the negligible signal observed in neat water, laser excitation of ZnPc in the presence of βγCD-NOPD shows the appearance of the characteristic triple−triplet absorption of ZnPc characterized by a maximum at 500 nm and a bleaching in the correspondence of the ground state absorption of the monomeric form at 680 nm (Figure S9).The triplet decay is biexponential with lifetimes τ 1 ∼ 20 μs and τ 1 ∼ 300 μs (Figure S10), which probably reflect different triplet population confined in different regions of the host.

βγCD-NOPD and Its
Ternary Assembly with LVB and ZnPc.Both LVB and ZnPc were co-encapsulated in βγCD-NOPD in two steps (see the Supporting Information).Figure 4A reports the spectrum of the supramolecular ensemble where the typical absorption of βγCD-NOPD at ca. 390 nm is accompanied by the characteristic absorption bands of LVB at ca. 240 nm and the partially monomerized ZnPc at ca. 680 nm.Note that the absorbance values, absorption maxima position, and spectral profile of all chromogenic components are basically not changed with respect to those observed when the guests are individually encapsulated in the polymeric host (compare Figure 4A with Figures 2 and 3).This finding indicates that the coencapsulation does not lead to a significant displacement or aggregation of the individual chromogenic components and is probably the result of their different affinity for the multiple binding sites of the polymeric host.Dynamic light scattering measurements (inset Figure 4) gave an average hydrodynamic diameter for the supramolecular assembly of ca. 15 nm.This value is slightly larger than that observed for the free host (∼11 nm), according to what has been already observed for similar branched polymers after the supramolecular encapsulation of multiple guests. 34,35In addition, the fluorescence properties of ZnPc when co-encapsulated with LVB were identical to those already found when it is individually entangled in the polymer, as confirmed by the same emission efficiency and fluorescence lifetime (Figure 4B and related inset).
The photodynamic properties of a multicargo nanoassembly were then investigated in terms of the capability to generate NO and 1 O 2 under visible-light excitation.
Irradiation of the polymeric nanoassembly with blue light induces bleaching of the main absorption band of the NOPD moiety at ca. 390 nm (Figure 5A), with a photolysis profile and photodecomposition rate (inset Figure 5A) very similar to  those observed for the free polymeric host (see Figure 1B and related inset for comparison) and accounting well for the NO release.Again, this was unambiguously confirmed by the direct NO detection (Figure 5B), which showed the photogeneration of NO occurring with the same efficiency as observed for the empty host βγCD-NOPD (see Figure 1C for the sake of comparison).These results suggest that the co-presence of LVB and ZnPc within the polymeric network does not induce either quenching phenomena or unexpected photochemical reactions competitive with the NO release.This is further confirmed by the lack of spectral changes observed in the correspondence of the absorption bands of LVB and ZnPc in the UV and red region, respectively (see Figure 5A), in accordance with the preservation of the structural integrity of these components after light irradiation.
Figure 6A shows the transient absorption spectra recorded after the initial laser pulse.The spectrum observed at 1 μs shows a maximum at 500 nm and bleaching at 680 nm, typical for the lowest triplet state of the monomeric form of ZnPc.The triplet absorbance is comparable to that observed in the absence of LVB (see Figure S9 for the sake of comparison).By taking into account the fact that the two solutions have the same absorbance at the excitation wavelength and that the molar extinction coefficient of the triplet state is not expected to significantly change, basically keeping their band profiles unchanged, the triplet absorbance can be directly related to the efficiency of the triplet population which, therefore is not affected by the co-presence of LVB.The chemodrug has no influence on the dynamic of the ZnPc triplet.The time evolution of this species with the elapsing time reveals in fact no additional transients formed concurrently to its decay (spectrum at 80 μs, Figure 6A), ruling out any possible bimolecular reaction.Analogously to what has been already observed in the absence of LVB (Figure S10), the ZnPc triplet decay was biexponential with lifetimes of τ 1 ∼ 18 μs and τ 2 ∼ 200 μs (Figure 6B).The triplet state is effectively quenched by oxygen as demonstrated by the significant shortening of the triplet decay, which, under air-equilibrated conditions, was  monoexponential with a lifetime of τ ∼ 10 μs (Figure 6C).This finding provides direct evidence that despite entanglement within the polymeric network, the triplet state of the PS is still accessible to molecular oxygen for the collisional energy transfer, which is crucial for 1 O 2 generation.
1 O 2 formation upon red-light excitation of the nanoassembly was unambiguously demonstrated by its diagnostic phosphorescence spectrum in the near-IR region with a maximum at ca. 1270 nm 9 (Figure 6D).A value for the 1 O 2 quantum yield Φ Δ = 0.25 ± 0.05 was obtained using methylene blue as a standard (see the Supporting Information).Note that the red-light irradiation under aerobic conditions does not lead to any significant changes in the whole absorption spectrum.This confirms not only the absence of any undesired bimolecular reaction of the triplet state of ZnPC with both the NOPD unit of the polymer and the co-encapsulated LVB but also rules out any potential oxidation of all chromogenic components by the photogenerated 1 O 2 .

CONCLUSIONS
We have synthesized a branched polymeric material covalently integrating both β and γCD units together with an NOPD within its macromolecular network.The photoresponsive βγCD-NOPD polymer is highly soluble in water, offers compartments with different sizes and hydrophobicities and represents a versatile polymeric host for the straightforward modular integration of multiple functional components.In fact, the simultaneous co-encapsulation of a hydrophobic chemodrug and a hydrophilic PS allows to achieve a system with multiple photo-chemotherapeutic cargos into a single supramolecular construct with functions that would be otherwise impossible to replicate with the separate components.
In particular, the polymeric host (i) amplifies the photoreleasing efficiency of the cytotoxic NO due to its active role as a reactant in the NO photorelease process; (ii) increases the solubility of LVB by more than 30-fold if compared with the free drug and more than 2-fold if compared with a similar polymer containing only βCD units; and (iii) disrupts the aggregation of the non-photoresponsive ZnPc, making it photochemically active and able to produce the cytotoxic 1 O 2 and to emit red fluorescence.
A remarkable point of this nanoconstruct is the absence of mutual interactions between the NOPD unit, the PS, and the chemodrug both in the ground and excited states, despite all components confined in the same host.This result is not trivial and allows the preservation of nature and efficiency of the individual photochemical properties of the phototherapeutic components while preserving the structural integrity of the chemodrug.In view of these results, we believe this polymeric multicargo nanoplatform, the first to our knowledge with the described peculiarities, may represent an intriguing system with potential trimodal photo-chemotherapeutic action to be tested in biological systems.

Scheme 2 .
Scheme 2. One-Step Copolymerization of the βCD Monomer Functionalized with Two NOPD Units and the Native γCD Using Epichlorohydrin as a Cross-Linker to Give the Mixed-Branched Polymer βγCD-NOPD a

Figure 1 .
Figure 1.(A) Normalized absorption spectra of βγCD-NOPD (solid line) and the model compound NOPD-1 (dotted line) in water.The inset shows the actual image of the water solution of βγCD-NOPD.(B) Absorption spectral changes observed upon light exposure of an air-equilibrated water solution of βγCD-NOPD (2 mg mL −1 ) at λ exc = 405 nm for time intervals from 0 to 16 min.The arrows indicate the course of the spectral profile with the illumination time.The inset shows the absorbance changes with the irradiation time at λ = 393 nm observed in air-equilibrated (○) and N 2 -saturated ( ■ ) solutions.(C) NO release profile observed for an aqueous solution of βγCD-NOPD (2 mg mL −1 ) (red line) and the model compound NOPD-1 (white line) upon alternate cycles of irradiation (λ exc = 405 nm) and dark.T = 25 °C.

Figure 3 .
Figure 3. Absorption spectra (solid lines) of ZnPc (10 μM) in water (a) and in the presence of βγCD-NOPD (2 mg mL −1 ) (b) and related fluorescence emission spectra (dotted lines) (λ exc = 640 nm) of samples a (c) and b (d).The inset shows the fluorescence decay and the related fitting of ZnPc in the presence of βγCD-NOPD recorded at λ exc = 635 nm and λ em = 690 nm.T = 25 °C.

Figure 4 .
Figure 4. (A) Absorption spectrum of the aqueous solution of βγCD-NOPD (2 mg mL −1 ) loaded with LVB (25 μM) and ZnPc (10 μM).The inset shows the hydrodynamic diameter and the actual image of the ternary supramolecular assembly.(B) Fluorescence emission spectrum of the sample reported in (A) (λ exc = 640 nm).The inset shows the fluorescence decay and the related fitting recorded at λ exc = 635 nm and λ em = 690 nm.T = 25 °C.

Figure 5 .
Figure 5. (A) Absorption spectral changes observed upon light exposure of an air-equilibrated water solution of βγCD-NOPD (2 mg mL −1 ) loaded with LVB (25 μM) and ZnPc (10 μM) at λ exc = 405 nm for time intervals from 0 to 19 min.The arrows indicate the course of the spectral profile with the illumination time.The inset shows the absorbance changes with the irradiation time at λ = 393 nm.(B) NO release profile observed for a sample as in (A) upon alternate cycles of irradiation (λ exc = 405 nm) and dark.T = 25 °C.
Synthesis and characterization of βγCD-NOPD and transient spectrum and time decay of the triplet state of ZnPc encapsulated in the polymer (PDF) ■ AUTHOR INFORMATION Corresponding Author Salvatore Sortino − PhotoChemLab, Department of Drug and Health Sciences, University of Catania, I-95125 Catania, Italy; orcid.org/0000-0002-2086-1276;Email: ssortino@unict.it