2019 Volume 2 Issue 6

INEOS OPEN, 2019, 2 (6), 185–195  Journal of Nesmeyanov Institute of Organoelement Compounds of the Russian Academy of Sciences Download PDF DOI: 10.32931/io1927r

Theranostic Agents for Photodynamic Therapy and Fluorescence Imaging Based on Organic Photosensitizers and Fluorophores

Abstract

The current review considers the examples of theranostic agents for photodynamic therapy (PTD) that have been described in the literature over the last decade. The problem of resonance energy transfer between the components of theranostic agents is addressed in detail. Special attention is paid to the authors' own works.

Key words: photodynamic therapy, fluorescence imaging, theranostic agent, photosensitizer, fluorophore, energy transfer.

 

1. Introduction

Nowadays, photodynamic therapy (PDT) is one of the most sparing and efficient methods for the treatment of oncological diseases [1, 2]. PDT is based on the administration of a solution of a photosensitizer (PS) that can selectively accumulate in cancer cells and, upon irradiation with the light of a certain wavelength, generate singlet oxygen and free radicals, which lead to tumor destruction [1–3]. PS emits a part of the energy of the absorbed light as fluorescence, which enables the definition of tumor localization. A combination of photodynamic therapy with fluorescence diagnostics (FD) enhances the efficiency of treatment in practice. Therefore, the development of systems for combined PDT and FD is an urgent scientific problem, which is explored by the modern discipline—theranostics. This field is receiving growing attention in addition to chemo- and radiotherapy [4, 5]. However, the number of reports devoted to the development of agents for theranostics in PDT is not so high. This review considers the examples of bifunctional molecules for PDT that are based on photosensitizers of a porphyrin series and different fluorophores. The photophysical processes that occur in these systems are analyzed to draw conclusions about the prospects for further progress in this area.

2. Theranostics in PDT

Currently, photosensitizers based on the following classes of tetrapyrrole macrocycles are used in practice and undergo clinical trials: porphyrins and their hydrated analogs (chlorins and bacteriochlorins), phthalocyanines, and metal complexes of these compounds. The structures of the mentioned compounds are based on a conjugated tetrapyrrole macrocycle, for which a change in the number of p-electrons on passing from porphyrins to bacteriochlorins significantly affects the position of PS absorption bands since it causes approaching of LUMO and HOMO levels and changes in the molecule symmetry [6]. Figure 1 depicts three PSs that refer to different classes of tetrapyrroles.

Figure 1. Chemical structures of selected porphyrin (porfimer sodium), chlorin (verteporfin),
and bacteriochlorin (redaporfin) used in clinical practice.

Porphyrin derivatives are characterized by long-wave absorption peaks in the range of 600–650 nm with low molar extinction coefficients [7]. The absorption spectra of chlorins, which lack one of the peripheral double bonds, show intensive signals in the range of 650–760 nm. Long-wave absorption maxima of bacteriochlorins are located in the range of 760–800 nm and are most appropriate for PDT, since the light of longer wavelengths can penetrate deeper into biological tissues (Fig. 2).

Figure 2. Penetration depths of light of different wavelengths into the skin. [E. Ruggiero et al., Dalton Trans., 2016, 45, 13012–13020.
DOI: 10.1039/C6DT01428C] – Published by The Royal Society of Chemistry

Fluorescence spectroscopy enables visualization of the processes in cells and measurement of the concentrations of biomarkers of pathological processes. With regard to PDT, fluorescence imaging helps to localize tumor sites and monitor treatment efficiency. The methods of optical imaging offer ample opportunities for investigation of biological objects; however, they stipulate the use of fluorophores that meet certain requirements. Figure 2 depicts schematically penetration depths of light waves into the skin [8]. The largest penetration depth into the skin is typical for near-infrared rays; furthermore, an important requirement is the location of an absorption maximum of a dye in the region of optical transparency of biological tissues, i.e., in the range of 650–850 nm, where blood components exhibit minimal absorption [6]. Therefore, the most promising fluorophores for practical application are those that absorb just in this range.

The application of tetrapyrrole PSs as diagnostic agents for PDT is associated with some problems. First of all, the excitation of a PS molecule using the low-intensity laser radiation for diagnostics will inevitably cause an accompanying photodynamic effect: the destruction of a part of tumor cells under the action of singlet oxygen; however, such an impact is undesirable since it can cause intensification of tumor growth. Secondly, own fluorescence characteristics of PS often do not meet the requirements of effective diagnostics. Most of PSs based on natural porphyrins feature low Stokes shifts, which complicates the detection of a fluorescence signal against scattered excitation light during fluorescence navigation [9, 10]. The third reason is that the compounds, which are used as photosensitizers, usually feature low quantum yields of fluorescence, since the main parameter that defines a possibility of their application as therapeutic agents is the high singlet oxygen quantum yield. Furthermore, the displacement of an absorption maximum to the longer wavelengths in the design of bacteriochlorin derivatives enhances the probability of the nonradiative relaxation of a molecule as a result of the internal conversion.

Taking into account the above-mentioned, the development of new agents that would combine the properties of therapeutic (photosensitizer) and diagnostic (fluorophore, FL) agents is a pressing challenge. The known examples of theranostic agents can be divided into two types by the coupling type of two functionalized moieties: covalent conjugates, in which FL and PS are connected through a spacer, and hybrid systems, which represent nanoparticles functionalized with FL and PS molecules. Next, we will consider both types of theranostics.

2.1. Conjugates of photosensitizers and fluorophores

Figure 3 demonstrates a functioning principle of a theranostic agent that represents a bis(chromophoric) system, in which functional fragments are connected through a spacer. The conjugate components must be chosen so that to ensure selective excitation of each of them; furthermore, they must not interact with each other in the excited state. In this case, a combination of two photoactive components in one molecule would enable selective treatment or detection of tumors by varying the excitation wavelength. The main problem in the development of conjugates for PDT is the probability of occurrence of a resonance energy transfer (RET), which impairs fluorescence characteristics of the system and prevents fluorescence diagnostics without a concomitant toxic effect.

Figure 3. Functioning principle of a theranostic agent based on a photosensitizer–fluorophore conjugate.

An important contribution to the development of theranostics for PDT was made by the group of Prof. R. Pandey, which is specialized in the elaboration of conjugates based on cyanine dyes [11–15]. Owing to the presence of a long chain of conjugated methine groups, cyanine dyes absorb in the near IR range. The drawbacks of this type of dyes include low Stokes shifts and a reduction in the photostability with an increase in the number of polymethine chain units, which shifts the dye absorption to the IR range [14]. One of the first examples of bis(chromophoric) systems for theranostics in PDT appeared to be a conjugate of highly efficient photosensitizer НРРН (a derivative of pyropheophorbide a, Fig. 4) [16] with a cyanine dye IR820 (compound 1a, Fig. 4) which features the absorption maxima at 632 and 836 nm [15, 17]. James et al. explored the spectral, photophysical and biological properties of a series of the cyanine chromophores; as a result, IR820 was chosen as the most promising dye for the application in theranostics [14]. Figure 5 shows the absorption spectra of the conjugate and its individual components in dichloromethane.

Figure 4. Structures of the conjugates containing IR-820 cyanine dye as a fluorophore.

The absorption spectrum of conjugate 1a corresponded in a large measure to a superposition of the spectra of НРРН and IR820 (Fig. 5), which implied weak interaction of the chromophores in the ground state. At the same time, the excitation of the photosensitizer in the conjugate composition with the light of 660 nm afforded a significantly lower-intensity emission signal than in the case of photoexcitation of individual HPPH. This indicates the energy transfer from the photosensitizer to the cyanine dye in the conjugate, which leads to the quenching of fluorescence of the donor component. Furthermore, an increase in the time of irradiation at the absorption maximum of PS afforded bleaching of the cyanine dye absorption band (830 nm, Fig. 5) with simultaneous enhancement of the photosensitizer fluorescence. These spectral changes testify the decomposition of the chromophoric system of IR820, which blocks the energy transfer from HPPH. The authors revealed that bleaching of the cyanine dye in the composition of the conjugate occurs only during the irradiation of the system at the absorption maximum of HPPH but not during direct photoexcitation of IR820, which indicates the decomposition of the fluorophore under the action of singlet oxygen formed in the system. In vivo studies with RIF tumor-bearing mice revealed the high selectivity of accumulation of 1a in invaded tissues and the possibility of obtaining fluorescence images of a tumor.

Figure 5. Absorption spectra of compounds IR-820, HPPH, and 1a in dichloromethane. In conjugate 1a, the irradiation of HPPH absorption at 660 nm caused a significant decrease in the absorption of the cyanine dye at 831 nm (a broad peak) and finally its complete photodestruction with a simultaneous increase in the intensity of HPPH absorption. (Reprinted with permission from Y. Chen et al., Bioconjugate Chem., 2005, 16, 1264–1274. DOI: 10.1021/bc050177o. Copyright (2005) American Chemical Society)

Using conjugate 1b with two photosensitizer moieties as an example (Fig. 4), it was shown [17] that the energy transfer from HPPH to CD leads not only to the quenching of fluorescence of the former but also to an essential reduction in the singlet oxygen quantum yield (Φ_Δ(HPPH) = 45%, Φ_Δ(1a) = 8%, Φ_Δ(1b) = 9%). This is caused by the fact that the deactivation of a singlet excited state of PS by the energy transfer to the cyanine dye prevails over the internal conversion to a triplet state, which takes part in subsequent interaction with a triplet oxygen form, resulting in singlet oxygen [18].

The effect of the length of a nonconjugated spacer on the degree of energy transfer and therapeutic efficiency of the conjugate was studied by the examples of 1с–1е [12] and 2а–2с [13] (Fig. 4). Interestingly, among compounds 1с–1е, the lowest degree of fluorescence quenching of the HPPH unit was observed in the case of conjugate 1d, which does not contain the longest spacer in this series. Moreover, this conjugate exhibited the highest singlet oxygen quantum yield (Φ_Δ= 6%, 11%, and 7% for 1с, 1d, and 1е, respectively). These facts suggest that the energy transfer in 1d proceeds less effectively than in the other conjugates. The authors explain this effect by the fact that the conformational flexibility of a methylene chain consisting of six units of conjugate 1e allows the chromophores to approach in space more effectively than in the case of a four-unit chain.

The stationary absorption and emission spectra of conjugates 2a–2c displayed the same regularities that were described earlier for 1a: a derivative of purpurinimide (PI, Fig. 4) serves as a donor in the energy transfer and its luminescence is considerably quenched, whereas a cyanine dye in the molecule composition undergoes photodestruction during prolonged irradiation. It is known that the efficiency of the energy transfer in a system strongly depends on the distance between interacting components. The distances between chromophores in the conjugates, calculated using the PM3 (SYBYL) method for the most extended conformation of each conjugate, composed 8.24 Å, 10.53 Å, and 16.58 Å for 2a, 2b, and 2c, respectively. However, in solution, each of the conjugates exhibits its own conformational distribution, and the distances between the chromophores can significantly vary. Williams et al. [13] calculated the contributions of different conformations to the conjugate structure in solution (Fig. 6). The average distances between the conjugate components were as follows: 8.18 (2а), 8.55 (2b), and 9.85 Å (2c).

Figure 6. Various conformers for conjugate 2a with limited flexibility in the linker are superimposed using the purpurinimide ring as a reference and examples of the low-energy conformers for conjugates 2b and 2c. (Reprinted with permission from M. P. A. Williams et al., Bioconjugate Chem., 2011, 22, 2283–2295. DOI: 10.1021/bc200345p. Copyright (2011) American Chemical Society)

The photodynamic efficiency of the conjugates was evaluated based on the half maximal inhibitory concentrations (IC₅₀) in Colon26 cells upon excitation at the absorption maximum of PI. The values of IC₅₀ were 0.22, 0.32, and 0.69 μmol/L for 2a, 2b, and 2c, respectively. The best activity was demonstrated by conjugate 2a featuring the shortest linker. As the linker between the chromophores elongates, the efficiency of therapeutic action reduces. The authors assume that this can be explained by the formation of the above-described conformers in cells. Compound 2a forms the conformers in which a purpurinimide moiety is open for the interaction with the surrounding, and singlet oxygen generated by it freely reaches cancer cells. In the case of 2b and 2c, the purpurinimide and dye moieties are located opposite to each other and this leads to the decomposition of the dye fragment in the conjugates under the action of reactive oxygen species.

In the in vivo experiments in mice bearing implanted Colon26 carcinoma, conjugates 2a–c demonstrated high selectivity of accumulation in cancer cells as well as good pharmacokinetic characteristics (in 48 h the skin phototoxicity disappeared). The highest fluorescence intensity and the fastest clearance from a tumor were displayed by conjugate 2b, whereas 2с fluoresced less intensively and the emission of 2a upon excitation of the cyanine dye (782 nm) appeared to be insufficient for efficient fluorescence diagnostics.

There are several examples of the conjugates of phthalocyanine photosensitizers with rhodamine fluorescent dyes [19, 20] that feature higher photostability than the cyanine fluorophores but display low Stokes shifts. Furthermore, their luminescence characteristics suffer from energy transfer. Thus, for example, the luminescence quantum yield of a rhodamine chromophore in the composition of conjugate 3 (Fig. 7) drops to 2% relative to Rh individual dye, which fluorescence quantum yield is 98% [20]. Grin et al. [21] described a triad consisting of indocyanine conjugated with two molecules of a photosensitizer based on bacteriochlorin, in which the energy transfer from the donor fluorophore component is realized with 95% efficiency.

Figure 7. Structures of the conjugates containing rhodamine and BODIPY dyes as fluorophores.

Over the recent decade, organic luminophores based on a BODIPY chromophoric system have been extensively used in bioimaging owing to the high photostability of many BODIPY derivatives, high molar extinction coefficients, and efficient fluorescence in the long-wave spectral region [22, 23]. It is important to note that the dyes based on BODIPY are often capable of sensitizing the generation of singlet oxygen [24, 25]. Recently, several examples of the conjugates for PDT based on BODIPY derivatives and phthalocyanine PSs have been reported [26–29].

The fluorophore and photosensitizer moieties in the composition of 4 are conjugated (Fig. 7). This leads to characteristic changes in the absorption spectra: both B and Q bands of the phthalocyanine moiety show significant bathochromic shifts upon conjugation with Ethynyl-BODIPY. However, even in the case of 4, the fluorophore emission is quenched to a large extent (Φ_F = 0.078 for 4 and 0.55 for nonconjugated Ethynyl-BODIPY). Göl et al. [26] assume that the photoexcitation energy transfer in conjugate 3 is realized by the exchange-resonance mechanism suggested by D. L. Dexter [30]. The singlet oxygen quantum yield for the conjugate appeared to be higher than that for the unsubstituted phthalocyanine (0.69 and 0.67, respectively), which can be explained by the synergistic effect of the conjugate components: the generation of ¹О₂ occurs not only by the phthalocyanine chromophore but also by the BODIPY derivative. In the case of conjugate 5, the excitation of the BODIPY unit gives rise to a fluorescence band with half the intensity than that for the free dye due to realization of the RET process; however, the singlet oxygen quantum yield for the conjugate remains at the same level as for the unsubstituted phthalocyanine (Φ_Δ = 0.56).

Of particular interest as fluorophores in theranostics are fluorescent dyes based on 1,8-naphthalimide derivatives. A chromophoric system of 1,8-naphthalimide is a popular optical platform for the creation of optical bleaching agents and dyes for polymer fibers [31, 32], electroluminescence materials [33, 34], fluorescent markers [35–37], and sensors for biological studies [38, 39]. The high photostability of fluorophores based on naphthalimide, the high values of Stokes shifts (over 150 nm in highly polar solvents), and the relative simplicity of chemical modification makes the luminophores based on 1,8-naphthalimide promising candidates for the application as fluorescent components of theranostic agents for PDT [40, 41].

Using the examples of conjugates of aminobacteriopurpurinimide and 4-methoxynaphthalimide 6a and 6b (Fig. 8), it was shown [41] that the efficiency of resonance energy transfer between the system components (EET_RET) in the case of the conjugate with a flexible linker (EET_RET = 50% for 6b) is essentially lower than that for 6a bearing a rigid spacer for which EET_RET = 96%. A distance between the chromophores obtained from the geometry optimized by the РМ6 (МОРАС) method composed 13.0 Å and 13.2 Å for 6a and 6b, respectively. The authors explain a significant difference in the efficiency of the energy transfer by the fact that the presence of a conformationally flexible spacer provides a possibility for the formation of a molecule conformer in solution, in which the chromophores would feature planar location and the energy transfer between them would be low-efficiency. At the same time, it is indicated that further elongation of a methylene chain could endow the conjugate with a propensity to form weekly fluorescing H-aggregates in solution stabilized by p-p stacking interaction of the chromophores.

;

Figure 8. Structures of bacteriopurpurinimide conjugates with 4-methoxynaphthalimide dye.

Recently we have published the results of investigation of the energy transfer in conjugates of a bacteriochlorin photosensitizer with fluorescent dyes based 1,8-naphthalimide 7a–c (Fig. 9) [42–44]. A derivative of the second generation photosensitizer bacteriochlorin e (compound BChl in Fig. 9) [45] was chosen as a photosensitizer component.

Figure 9. Structures of bacteriochlorin–naphthalimide conjugates 7a–c and monochromophores NIa, NIb, and BChl.

Besides the Soret band at 400 nm, the absorption spectra of the bacteriochlorin derivatives show intensive long-wave bands with the maxima at 750 nm, which fall into the therapeutic window of biological tissues. Furthermore, the derivatives of bacteriochlorin feature low dark toxicity, rapid clearance from the organism, and low skin phototoxicity. Conjugates 7a–c were synthesized by 1,3-dipolar cycloaddition of a propargyl derivative of bacteriochlorin to naphthalimide dyes bearing azide functional groups. The optical characteristics of these conjugates are considered below by the example of compound 7а [42]; the absorption and fluorescence spectra of 7b and 7с feature similar regularities.

Figure 10 depicts the electronic absorption and fluorescence spectra of compounds 7a–c, monochromophores NIa and BChl, and their equimolar mixtures. The absorption spectrum of the conjugate strongly resembles the spectrum of the equimolar mixture of NIa and BChl (Fig. 10c), which points to the weak interaction of the chromophores in the ground state. The absorption maximum of the naphthalimide chromophore in the conjugate composition undergoes an insignificant bathochromic shift relative to the spectrum of individual dye NIa. This can be explained by the acceptor effect of a triazole moiety, which appearance in the conjugate structure enhances the ICT process compared to N-butylnaphthalimide NIa. The fluorescence maxima of the dye and bacteriochlorin are at 760 nm (Fig. 10b) however, the fluorescence spectrum of the dye represents a wider band that falls into the IR region. In the case of the equimolar mixture (Figs. 10c,d), the excitation at 490 nm, which is absorbed mainly by the dye, gives rise to a broad emission band with the shape resembling the fluorescence spectrum of the individual dye, albeit with a low-intensity sharp peak with the maximum at 760 nm which corresponds to fluorescence of bacteriochlorin as a result of direct photoexcitation.

Upon irradiation of the conjugate bearing two covalently bound photoactive components with the light of 460 nm, the observed fluorescence spectrum drastically differs from that of the equimolar mixture: a narrow fluorescence peak appears at 760 nm that corresponds to fluorescence of free bacteriochlorin, whereas fluorescence of the naphthalimide dye is quenched completely (Fig. 10d). This evidences that the naphthalimide fluorophore, after the transition to the excited state, takes part in a rapid nonradiative process of intramolecular energy transfer from the naphthalimide donor moiety to the bacteriochlorin acceptor fragment.

Figure 10. UV/Vis absorption (a and c) and fluorescence emission (b and d) spectra of compounds BChl, NIa, 7a, and the equimolar mixture of BChl and NIa (denoted as "BChl + NIa") in acetonitrile. The excitation wavelength was 460 nm for NIa, 7a, and BChl + NIa and 515 nm for BChl. The concentrations of all compounds were 4.7 10^–6 M. [P. A. Panchenko et al., Phys. Chem. Chem. Phys., 2017, 19, 30195–30206. DOI: 10.1039/C7CP04449F] – Reproduced by permission of the PCCP Owner Societies

The nature of photophysical processes that occur in conjugate 7a during photoexcitation was studied by transient absorption spectroscopy (TRABS).

Figure 11а depicts the TRABS spectral map for conjugate 7a. A positive signal (red region) corresponds to the absorption of the excited state of 7a; a negative signal (blue region) may correspond to either stimulated emission or the so-called ground state bleaching of the chromophore.

Figure 11. TRABS spectral map with subpicosecond time resolution (a) and TRABS kinetics at 750 nm (b) of 7a in acetonitrile. The excitation wavelength was 470 nm. [P. A. Panchenko et al., Phys. Chem. Chem. Phys., 2017, 19, 30195–30206. DOI: 10.1039/C7CP04449F] – Reproduced by permission of the PCCP Owner Societies

The spectral time map depicted in Fig. 11a exhibits a negative signal with the maximum at 750 nm. Since this region is characteristic of the absorption band of the bacteriochlorin chromophore (see Fig. 10a), this negative signal can be attributed to the ground state bleaching of bacteriochlorin, which suggests the transition of the photosensitizer moieties to the excited state. The positive signal in the range of 500–680 nm is likely to correspond to the absorption of S₁ states of both of the chromophores. At 750 nm, this signal can also possess the nonzero intensity and reduce with an increase in the delay time, which explains the appearance of the increasingly negative signal in Fig. 11b even after 4.1 ps—the characteristic relaxation time of the excited state of bacteriochlorin.

Based on the time profile at 750 nm (Fig. 11b), the characteristic time of the photosensitizer transition to the excited state was calculated that composed 0.53 ps. Using the characteristic time of the photosensitizer transition to the excited state, the rate constants   (k_RET = 1 / τ_RET = 1 / 0.53∙10^-12= 1.89∙10¹² s^–1) and efficiency of the RET process were calculated; the latter was found to be 99.9%.

The efficiency of the energy transfer in the conjugates was also estimated based on the theoretical calculations within the resonance-induced Förtser model. The theoretical efficiency of the energy transfer and the rate constants of the RET process in the conjugates were calculated using relations (1) and (2) [30]:

	(1)
	(2)

where τ_D is the lifetime of the excited state of the donor chromophore (naphthalimide) in the absence of the acceptor (bacteriochlorin) and r is the distance between the donor and the acceptor. The distance between the donor and the acceptor (r) was found by optimization of the molecule geometry using the PM6 method. R₀ is the Förster critical radius (the distance between the chromophores at which the efficiency of the energy transfer is equal to 50%). The experimental values of the energy transfer in conjugate 30 were evaluated using relation (3):

	(3)
	(4)

where φ^fl  and φ_D^fl are the quantum yields of the donor chromophore in the presence and absence of the acceptor, τ_D and τ are the fluorescence lifetimes of the donor in the presence and absence of the acceptor, respectively. Since the quantum yields of the donor components of compounds 7a and 7b in the presence of acceptors cannot be measured, the calculation was carried out by formula (4) using the lifetimes. Table 1 lists the main characteristics of the energy transfer in conjugates 7a–c; the efficiency of the energy transfer in all the bis(chromophoric) systems was close to 100%. 

Table 1. Photophysical characteristics of compounds BChl and 7a–c in acetonitrile

	φfl	λflmax (λex), nm	EETRET, %
			Theor.	Exp.a
BChl	0.030	758 (515)	–	–
7a	0.023	755 (460)	97.63	99.9
7b	0.028	757 (470)	99.79	b
7c	0.021	753 (470)	6.50	93.0

  ^a calculated using the fluorescence lifetime of the conjugate donor component;
  ^b the efficiency of the energy transfer is difficult to calculate.

As can be seen from formula (2), the value of the rate constant for the energy transfer in the system reduces proportionally to the distance between the chromophores (r) to the sixth power. To increase the distance between the chromophores and to minimize the energy transfer, a polyglycol spacer was introduced into the composition of conjugate 7c (Fig. 9) [43]. The geometry of conjugate 7с was optimized by the PM6 method using МОРАС software. The calculated distance between the chromophores in the conjugate was found to be 48.0 Å. Using the critical value of the Förster radius obtained from the spectral characteristics of monochromophores, the theoretical efficiency of the energy transfer in conjugate 7с was calculated and composed 6.5%.

Figure 12 depicts the electronic absorption and fluorescence spectra of compounds 7b, 7c and the equimolar mixture of BChl and NIb. In the case of the equimolar mixture, the excitation at 470 nm, which is absorbed mainly by the naphthalimide dye, leads to the appearance of a broad emission band corresponding to the fluorescence of the individual dye and a low-intensity peak at ca. 760 nm corresponding to the fluorescence of bacteriochlorin. The irradiation of 7b and 7c with the light of 470 nm affords a narrow fluorescence peak with the maximum at 760 nm, which corresponds to the fluorescence of bacteriochlorin, whereas the fluorescence of the naphthalimide dye is quenched (Fig. 11b). However, in the case of conjugate 7c, the intramolecular energy transfer proceeds less effectively than in the case of 7b. The degree of the energy transfer in conjugate 7c was estimated using the lifetimes of the naphthalimide chromophore in the presence and absence of the acceptor according to formula (4) and composed: 1 – (0.065/0.93) = 0.93 (93%).

Figure 12. UV/Vis absorption (a) and fluorescence emission (b) spectra of compounds 7b and 7c and the equimolar mixture of BChl and NIb (denoted as "BChl + NIb") in acetonitrile. The excitation wavelength was 470 nm. The concentrations of all compounds were 5∙10^–6 M [44].

A significant difference in the efficiencies of the RET process derived from the theoretical and experimental data can be explained by the fact that the polyglycol spacer of 7c forms predominantly a coiled conformation in solution, which provides efficient approaching of the chromophores in space. To reduce the energy transfer efficiency, it is necessary to introduce a spacer that has a more rigid structure in solution. The energy transfer in conjugates 7b and 7c effectively proceeds in biological media: in a solution of bovine serum albumin and rabbit blood serum.

The efficiency of conjugate 7a was studied on human lung adenocarcinoma cells А549 [42]. Upon irradiation of the cells incubated with the conjugate solution at the absorption maximum of the fluorophore (488 nm), the excitation energy is efficiently transferred from the naphthalimide moiety to the bacteriochlorin acceptor, which leads to the appearance of a fluorescence signal of the latter (760 nm) as well as the formation of reactive oxygen species (Figs. 13a,c). During prolonged highly intensive irradiation, a photodynamic effect is observed in cells: the decomposition of the cell components and the bacteriochlorin chromophore under the action of reactive oxygen species (Figs. 13b,d).

Figure 13. Intracellular distribution of 7a fluorescence in A549 cells. The distribution of fluorescence in the ranges of 550–650 nm (a and c) and 730 nm (b and d) at the low-power excitation 488 nm before (a and b) and after (c and d) the high-power prolonged illumination (photobleaching) of the cells with 488 nm wavelength. The cells were incubated with 2 mM of 7a for 3 h. [P. A. Panchenko et al., Phys. Chem. Chem. Phys., 2017, 19, 30195–30206. DOI: 10.1039/C7CP04449F] – Reproduced by permission of the PCCP Owner Societies

The partial decomposition of the acceptor fragments leads to the manifestation of the own fluorescence by the naphthalimide dye (Fig. 13b). The fluorescence response of the naphthalimide chromophore can be used in practice to select optimal doses and intensities of irradiation in PDT. Compound 7a displays photodynamic activity towards А549 cell line. The irradiation in the range of long-wave absorption maximum of bacteriochlorin leads to the death of about 90% of cells at the concentration of 0.7 mmol/L. In the concentrations explored (<8 μmol/L), the conjugate does not exhibit toxicity for the cells without photoexcitation.

Using the method of a chemical trap, the singlet oxygen quantum yields were calculated for conjugates 7а–с. This method is based on the addition of a singlet oxygen trap to a photosensitizer solution, namely, 1,3-diphenylisobenzofuran (DPIBF) that quantitatively reacts with ¹O₂ particles, resulting in an endoperoxide, which is not colored in the visible range [46, 47]. The quantum yields presented in Table 2 allow one to draw an important conclusion: the conjugation of the naphthalimide fluorophores with bacteriochlorin in the case of 7a–c does not lead to a reduction in the efficiency of singlet oxygen generation by the photosensitizer.

Table 2. Singlet oxygen quantum yields for BChl and 7a–c in acetone

Compound	BChl	7a	7b	7c
ΦΔ	79%	82%	67%	84%

It can be concluded that the main problem during the development of bis(chromophoric) systems is the possibility of realization of the energy transfer between the chromophores, which deteriorates the fluorescence properties of a fluorophore or a photosensitizer, and, furthermore, makes impossible the diagnostics without a concomitant toxic effect. The creation of conjugates for theranostics requires a search for efficient methods for controlling the processes of energy transfer, such as the introduction of spacers that can be cleaved under the action of some or other conditions or long spacers that possess low conformational mobility.

2.2. Functionalized nanoparticles for theranostics in PDT

As it was already mentioned, an alternative approach to the covalent binding of a fluorophore and PS in a conjugate composition is the production of hybrid nanoparticles which surface is modified with chromophore molecules [48]. Nowadays, new PDT agents based on the modified gold [48] and silicon dioxide [49] nanoparticles as well as quantum dots [50, 51] and upconversion nanoparticles [52, 53] are actively developed. To keep a reasonable length of the present review, we will consider the modification only with upconversion nanoparticles (UCNPs). Their apparent advantage is the possibility of creation of systems that can be excited with IR light.

It is well known that most of organic luminophores emit luminescence of the higher wavelength than the absorbed light, i.e., possess positive Stokes shifts [54]. Owing to considerable progress in the field of development of ultrashort pulsed lasers over the last decades, the branch of physics that studies nonlinear optical processes arising in luminophores under the action of high-intensity radiation is actively developing now. One of these processes is the upconversion of photon energy—the process of transformation of several photons of the lower energy (or the longer wavelength) to one photon of the higher energy (the shorter wavelength) [55]. This process attracts continuous interest of researchers who work in the field of fluorescence visualization, since it allows one to obtain a fluorescence response in the visible region using IR irradiation for excitation, which is characterized by the highest depth of penetration into biological tissues. The modification of upconversion nanoparticles is one of the most promising trends in the development of new agents for fluorescence imaging [5, 56, 57]. These particles represent nanoparticles of an inert inorganic material doped with rare-earth elements, which enable the realization of the upconversion of the energy of absorbed radiation [55].

The group of Prof. J. Perez-Prieto from the University of Valencia obtained sodium tetrafluoroyttriate (NaYF₄) nanoparticles doped with trivalent lanthanides (erbium, ytterbium, and thulium) [58]. A principle of upconversion is illustrated in Fig. 14. The ytterbium ion absorbs light radiation at the wavelengths of 975–980 nm; its excited state is able to act as a donor during energy transfer to the erbium and thulium ions, which results in the realization of the electron transfer to the energies corresponding to the excited states of Er³⁺ and Tm³⁺ ions.

Figure 14. Upconversion mechanism of the lanthanide UCNPs codoped with Yb³⁺ and Er³⁺ or Yb³+ and Tm³⁺. (Reprinted with permission from J. Zhou et al., Chem. Rev., 2015, 115, 395–465. DOI: 10.1021/cr400478f. Copyright (2015) American Chemical Society)

The energy states of the acceptor ions possess long lifetimes; therefore, the donor ion can transfer nonradiatevely several photons to the long-living acceptor state. Hence, the excited states of Er³⁺ and Tm³⁺ relax to the ground state emitting the photons of the higher energy than the initial exciting radiation [55].

An idea of the application of upconversion particles for PDT consists in the realization of the energy transfer from the upconversion nanoparticle NaYF_4:Yb,Er,Tm to a photosensitizer for the generation of ¹O₂ upon excitation of the former with IR light, whereas the shorter wave channel serves for excitation of only naphthalimide fluorophore and production of a fluorescence response (Fig. 15). To realize a series of photophysical processes on the surface of a nanoparticle, it is necessary for a fluorophore, a photosensitizer, and a NaYF_4:Yb,Er,Tm particle to correspond to each other by the optical characteristics.

Figure 15. Functioning principle of hybrid nanoparticles for PDT and structures of the photosensitizer PP and fluorophore NI immobilized on the surface of the nanoparticles. (Reprinted with permission from L. Francés-Soriano et al., Chem. Mater., 2018, 30, 3677–3682. DOI: 10.1021/acs.chemmater.8b00276. Copyright (2018) American Chemical Society)

Upon excitation with the wavelength of 975 nm, the hybrid NaYF₄ nanoparticles doped with ytterbium, erbium and thulium ions demonstrate narrow luminescence peaks with the maxima at 475, 520, 540, 650, and 808 nm as a result of the effective upconversion process [58]. We chose N-aminobacteriopurpurinimide PP (Fig. 15) as a photosensitizer for grafting on the surface of NaYF_4:Yb,Er,Tm. Its long-wave absorption peak overlaps with the particle fluorescence in the range of 808 nm (Fig. 16) [41], which enables energy transfer between these components. A derivative of 4-pyrazolinyl-substituted naphthalimide NI bearing a terminal carboxy group was used as a fluorophore. It features a broad absorption band in the range of 490 nm and orange luminescence with the maximum at 637 nm.

Figure 16. Luminescence spectra of NaYF_4:Yb,Er,Tm/NI (red line) and unmodified NaYF_4:Yb,Er,Tm (black line), excitation at 975 nm (a); absorption spectra (dash line) and fluorescence spectra (solid line) of NaYF_4:Yb,Er,Tm/NI/PP, excitation at 490 nm (b); emission spectra at λ_ex = 975 nm for a dispersion of NaYF_4:Yb,Er,Tm (black line) and NaYF_4:Yb,Er,Tm/PP/PEG in water (red line) normalized at 650 nm (c); emission spectra normalized at 650 nm for dispersions of NaYF_4:Yb,Er,Tm (dash line) and NaYF_4:Yb,Er,Tm/NI/PP (solid line), excitation at 975 nm (d). The concentration of NP in solution was 1g/L, the solvent was acetonitrile. (Reprinted with permission from L. Francés-Soriano et al., Chem. Mater., 2018, 30, 3677–3682. DOI: 10.1021/acs.chemmater.8b00276. Copyright (2018) American Chemical Society)

To study the effect of immobilization on the optical properties of the photoactive components, NaYF_4:Yb,Er,Tm nanoparticles modified solely with the molecules of PP (herein, NaYF_4:Yb,Er/PP) and solely with NI dye (herein, NaYF_4:Yb,Er,Tm/NI) were obtained. The hybrid nanoparticles, which surface was modified with the molecules of NI and PP, are further denoted as NaYF_4:Yb,Er,Tm/NI/PP. Using the molar extinction coefficients of NI and PP in acetonitrile, it was established that on average there are 325 molecules of NI and 5789 molecules of PP on the surface of a single nanoparticle.

From the fluorescence spectra presented in Fig. 16a,b, it is obvious that the excitation of a solution of NaYF_4:Yb,Er,Tm/NI and NaYF_4:Yb,Er,Tm/PP particles at 975 nm leads to a reduction in the intensity of green luminescence of erbium and thulium in the range of 520–550 nm (by 80% and 55%, respectively) compared to the unmodified NaYF_4:Yb,Er,Tm particles and the appearance of low-intensity bands in the range of 550–600 nm (Fig. 15a) and 830 nm (Fig. 15b), which correspond to the emission bands of NI and PP fluorophores. This spectral effect testifies that the photoexcitation energy on the particle surface is transferred not only to photosensitizer PP but also to the naphthalimide dye. The irradiation of NaYF_4:Yb,Er,Tm/NI/PP particles with the light of 975 nm (Fig. 16d) also leads to quenching of luminescence in the green region (by 40%) and the appearance of an intensive fluorescence band with the maximum at 830 nm, which corresponds to the emission of N-aminobacteriopurpurinimide.

Interestingly, the intensity of fluorescence of purpurinimide upon irradiation of NaYF_4:Yb,Er,Tm/NI/PP hybrid particles is essentially higher than that observed upon irradiation of the particles without a fluorophore. This is connected with the fact that several energy transfer processes are realized simultaneously on the surface of NaYF_4:Yb,Er,Tm/NI/PP particles. The photoexcitation of yttrium (975 nm) is transferred to the erbium and thulium ions; then, it is transferred to the photosensitizer and fluorophore molecules; the photoexcitation of the fluorophore is followed by the energy transfer to PS, which leads to enhancement of its fluorescence. Hence, fluorophore NI serves as an antenna which strengthens the emission of a photosensitizer upon excitation of the particles at 975 nm.

The excitation of NaYF_4:Yb,Er,Tm/NI/PP particles in the range of 490 nm (Fig. 16c) leads to the appearance of a broad band with the maximum at 637 nm, which corresponds to the naphthalimide fluorophore. This suggests that the undesired energy transfer from the naphthalimide to the photosensitizer is suppressed on the surface of nanoparticles to a certain extent. The integral of overlapping of the fluorescence spectrum of the naphthalimide fluorophore and the absorption spectrum of N-aminobacteriopurpurinimide is a great magnitude: 8.79∙10¹⁴ M^–1∙m^–1∙nm⁴. The critical Förster radius, i.e., the distance at which the efficiency of energy transfer between the chromophores composes 50%, for the pair of chromophores under consideration comprises 31 Å, whereas the average diameter of NaYF_4:Yb,Er,Tm/NI/PP nanoparticles composes 23 nm (230 Å). It can be assumed that, in the case of the nonuniform distribution of NI and PP molecules over the surface of nanoparticles, the energy transfer process will efficiently proceed not for all fluorophore pairs, the rest will provide a fluorescence response in the range of 637 nm owing to the incomplete energy transfer.

Figure 17 shows the images of cells incubated with a solution of the nanoparticles (100 μg/mL), which were obtained using confocal fluorescence microscopy. The excitation of cells with the light of 975 nm leads to the emission of both of the particles themselves (Fig. 17а) and the naphthalimide moiety (Fig. 17b).

Figure 17. Confocal microscope images of SH-SY5Y cells incubated with UCYbErTm/NI/PP: λ_ex = 975 nm, λ_em = 515−580 nm (a); λ_ex = 975 nm, λ_em = 590−650 nm (b); overlap of A and B (yellow) (c); λ_ex = 488 nm, λ_em = 570−670 nm (d). Scale bar: 50 μm. (Reprinted with permission from L. Francés-Soriano et al., Chem. Mater., 2018, 30, 3677–3682. DOI: 10.1021/acs.chemmater.8b00276. Copyright (2018) American Chemical Society)

The naphthalimide chromophore NI absorbs in the range of 490 nm. It can be excited with two photons of the near-infrared range (880 nm). Two-photon photoexcitation allows one to carry out fluorescence diagnostics without a concomitant therapeutic effect, since the particles are transparent in the range of 880 nm (Fig. 17a). Evaluation of the photodynamic efficiency of NaYF_4:Yb,Er,Tm/NI/PP nanoparticles on a human neuroblastoma cell line SH-SY5Y showed that the hybrid nanoparticles efficiently generate singlet oxygen in cell culture and affect cancer cells. The cell viability in a control experiment (irradiation for 7 min without preliminary incubation with a solution of the theranostic agent) composed 99.18%, which allows one to reliably associate this effect with the photodynamic action of the nanoparticles explored. The resulting nanohybrids exhibited dark toxicity during 24 h incubation at the concentrations above 500 μg/mL; the experiments were carried out using 100 μg/mL solution.

Hence, the hybrid nanoparticles functionalized with N-aminobacteriopurpurinimide and fluorophore NI are effective theranostics agents. They enable both fluorescence imaging of cancer cells without a concomitant toxic effect and photodynamic therapy with simultaneous fluorescence navigation.

4. Conclusions

The analysis of the reported data on the development of theranostic agents for photodynamic therapy showed that a photosensitizer and a fluorophore combined in the structure of a single molecule interact in the excited state: the photoexcitation energy transfer is realized between them that leads to deterioration of the photodynamic or fluorescence properties of the conjugate. To suppress this process, spacers must be introduced that can provide efficient separation of the chromophores in space. The immobilization of upconversion nanoparticles on the surface is one of the promising routes for conjugation of the functional components of a theranostic agent into a single system since the resulting conjugates enable diagnostics without concomitant toxicity.

Acknowledgements

This work was supported by the Russian Foundation for Basic Research, project no. 16-13-10226.

Absorption and luminescence spectroscopic studies were performed with the financial support from the Ministry of Science and Higher Education of the Russian Federation using the equipment of the Center for Molecular Composition Studies of INEOS RAS.

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