Drug-delivery and multifunction possibilities of hypocrellin photosensitizers

Photodynamic therapy (PDT) has been a routine treatment of tumors and some microvascular diseases, but clinically available photosensitizers are still scarce. Among all kinds of photosensitizers, hypocrellins possess the most characteristics of ideal photosensitizers, such as, high photo-activity but low dark toxicity, fast clearance from tissues. This review is focused on two main topics, drug-delivery problem of hypocrellins and how the environment-sensitive °uorescence of hypocrellins was used for recognition of various biomolecules. Drug-delivery of hypocrellins was mainly achieved in two strategies— preparing the drug-delivery vehicles and ̄nding quantitatively amphiphilic derivatives. Hypocrellin °uorescence originated from the intramolecular proton transfer is very distinct from other kinds of photosensitizers. Recently, it was proved that quantitative hypocrellin °uorescence could not only recognize various biomolecules, including proteins, polysaccharides and lipids, but also distinguish the speci ̄c binding from nonspeci ̄c binding with some kind of biomolecules. Meantime, hypocrellin °uorescence was pH-sensitive. It is known that tumor cells or tissues have the features of a large amount of lipid, neonatal collagen, over-expression of polysaccharides, and lower pH values compared to normal tissues. According to the relative but not absolute speci ̄city, further studies on quantitative recognition of various biomolecules at a cellular level, may ̄nd a new clue to treat tumors by joint usage of photodynamic diagnosis (PDD) and PDT.


Introduction
Photodynamic therapy (PDT) has become a regular methodology to treat various tumors clinically, 1 and recently, it has been successfully used to treat some microvascular diseases, such as port wine stains (PWS), age-related macular degeneration (AMD). 2,3t is known that light, photosensitizer and oxygen are the three primary factors for PDT but photosensitizer is the key important one.An ideal photosensitizer should possess high PDT activity but low dark toxicity as well as fast clearance from tissues, in addition, it should be readily delivered but not seriously sacri¯ce the PDT activity in vivo.Besides the photosensitization activity to produce reactive oxygen species, the photosensitizers are °uorescent.It has long been hoped that a photosensitizer might be used for photodynamic diagnosis (PDD) and PDT at the same time, 4 which may be a good strategy to conquer cancers, however, lack of speci¯c target and °uorescence signal prohibit the program.
6][7] However, their low absorption in the phototherapeutic window (600-900 nm) makes them not suitable for PDT of solid tumors. 8In addition, hypocrellins are lipophilic organic compounds, which promotes the cellular uptake 9 but prohibits the drug-delivery and bioavailability. 10esides PDD depends on the typical °uorescent signals of a photosensitizer on the targets.It was indicated that some biological molecules were over expressed in tumor cells or tissues, 11,12 however, they are quantitatively but not qualitatively speci¯c relative to normal cells.Therefore, tumor cells or tissues may not be recognized by non-speci¯c °uorescent information.It was known that hypocrellin °uorescence was originated from intramolecular proton transfer 13 and sensitive to solvents. 14In this review, some recent progresses of researches on the drug-delivery and environment-sensitive °uorescence of hypocrellins are concerned.

Drug-Delivery of Hypocrellins 2.1. Drug-delivery vehicles
One important strategy for clinical application of lipophilic photosensitizers is to prepare drug-delivery vehicles in nanometer sizes with biocompatible materials, which are water soluble and readily control drug-delivery in vivo.

Liposomal hypocrellins
The liposome is composed of lipid bilayers selfassembled naturally or arti¯cially by phospholipids.The liposome has good biocompatibility, targeting, controllable rates of drug e®lux, and has been widely employed as vehicles for lipophilic drugs, for instance, vertepor¯n, a PDT drug for AMD, is clinically used in a liposomal preparation. 15Ultrasonic method was previously used to prepare the liposome of HA and HB which can retain about 70% of PDT activity of HA or HB. 16,17A pharmacokinetic study in model animal indicated that the liposomal HA had much higher accumulation in tumor tissues than HA in DMSO, suggesting that the liposome formulation could preferentially target tumor tissues. 18Photosensitization of liposomal HB produced superoxide anion radicals, hydroxyl radicals and singlet oxygen, suggesting both Types I and II photosensitization mechanisms involved. 16ecause liposome in solution is not stable, solid liposome was prepared by reverse evaporationlypophilization drying. 19More recently, liposomal hypocrellin powder was prepared by high pressure homogenization method. 20This method not only promoted the drug loading up to 1 mg/mL, but also got rid of tween 80 which was not recommended by pharmacopoeias, in addition, the liposome size was distributed uniformly and controllably.Taking chicken combs as models of PWS, it was proved the liposomal hypocrellin B could selectively destruct the dermal microvasculatures, but not hurt overlying epidermis with a HB dose of 0.5-1 mg/kg and a light dose of 120 J/cm 2 , suggesting the safety and e±ciency.

Nanomicelles of hypocrellin
A micelle is prepared by the use of surfactant molecules to load lipophilic drugs.3][24] It was reported that triton X-100 micelles of HB could maintain most of the photosensitization activity, however, the sizes of micelles are too large to be used as PDT drugs.But this formulation may be used as photodynamic pesticides.

Nanoemulsion of hypocrellin
The emulsion is a preparation of heterogeneous system formulated by two or more liquids which are not inter-solvable.Generally, the particle sizes of currently clinically used emulsions are in micrometer level, which is too large to be used as PDT drugs.Recently, the hypocrellin emulsion was prepared in the sizes distributed from 30 to 60 nm 25 with a drug loading of 1 mg/mL, which is suitable for use in PDT.In addition, the PDT activity of the emulsion preparation is the highest among all the preparations.The hypocrellin emulsion is readily prepared with low-cost materials, however, the storage time is no more than six month, which is too short to satisfy the quality standard of a new drug.

Nanoparticles of hypocrellins with bio-materials
Nanoparticles are known as nanospheres or nanocapsules, which carry drugs inside or outside particles by physical, chemical adsorption or even covalent combination.Previously, hypocrellin nanoparticles were prepared with gelatins or polysaccharides in the sizes of about 100 nm. 26,27The preparation is not only drug-delivered but also remains most of the PDT activity.Until now, many kinds of hypocrellin nanoparticles were reported, including HA-Silica nanoparticles, nanoscale porous ceramic nanoparticles, pH-responsive silica nanoparticles, TiO 2 nanoparticles, hierarchical gold/ copolymer nanostructures or lipid-coated gold nanocages, etc. [28][29][30][31][32][33] In vitro studies showed most of these formulations remained most of the PDT activity of their parents, but whether they can be used in vivo is still questionable.The formulations are usually very stable due to presence of some hardeners, however, controlled-release drugs may be appropriate for PDT of solid tumors, but not for PDT of microvascular diseases.

Chemical modi¯cations of hypocrellin structures
Aqueous solubility is essential for both drugdelivery and bioavailability, 34 but lipophilicity is also necessary for cellular uptake which is closely related to PDT activity in vivo.Generally, a lipophilic photosensitizer exhibits high cellular uptake which is necessary for PDT activity in vivo, but its self-aggregation may lead to a risk of vascular embolization.On the other hand, a hydrophilic photosensitizer is ready for drug-delivery, but its little cellular uptake leads to low PDT activity.For about 20 years, chemical modi¯cations of hypocrellins were focused on improving the aqueous solubility, however, the improvement had only a relative signi¯cancethe solubility of some derivatives was somewhat higher than their parents (several M level), but it was either too low to satisfy clinical requirement for drug concentration, or too high to have a cellular a±nity.Consequently, in a viewpoint of clinical requirement for drugdelivery, an ideal derivative should have a quantitative instead of qualitative amphiphilicity, i.e., the lipophilicity should be kept as large as possible as long as the aqueous solubility satis¯es the clinically required concentration of the drug. 34Generally, clinically required concentration (C) could be estimated according to the following equation 35 : In the equation, D HB is 0.5 mg/kg based on the animal experiments for PDT of port wine stains (PWS) 36 ; W is an average weight of a person, 80 kg; V is commonly acceptable volume for one time intravenous injection, 30 mL and A HB /A Derivative is ratio of PDT activity of HB to the derivative.So if the PDT e®ect of a hypocrellin derivative is equal to that of HB, the minimal concentration for directly intravenous injection is calculated to be 1.3 mg/mL, which indicates the threshold for the clinically required solubility.

Hypocrellin-metal ion complexes
Hypocrellins facilely react with aluminium(III), magnesian(II), zincl(II) or lanthanum(III) ions to form water-soluble complexes.][39] Generally, the molecular weight of the complexes was uncertain perhaps due to formation of polymerlike structures. 371][42] An in vivo study indicated the hypocrellins complexes with aluminium ions might be dissociated in vivo, then lead to aggregation of free hypocrellins and vascular embolization (unpublished result), which may be a common problem for this kind of complexes.

Introduction of a polar substituent
Introduction of a polar substituent to a lipophilic molecule is a common strategy to improve the aqueous solubility.Sulphonate, glycosylate, cyclodextrin and dicysteine substituted hypocrellin derivatives [43][44][45][46][47] were easily soluble in aqueous solution and exhibited some photosensitization activity in vitro, but they almost lost the PDT activity in vivo due to low cellular uptake. 48Previously, a theoretical method was developed to evaluate the amphiphilicity of hypocrellin derivatives and a concept of quantitative amphiphilicity was proposed. 49Evaluated based on this idea, mercapto, amino-acid, ethanolamine, morpholine, piperazine and dipeptide substituted hypocrellin derivatives exhibited higher solubility than their parents, [50][51][52][53][54] but the solubility was lower than the threshold of clinically required concentration as mentioned above.6][57] It is well known that surfactants are theoretically amphiphilic.However, surfactant-like HB derivatives, sodium 12-2-HB-aminododecanoate and sodium 11, 11 0 -5,8-HB-dimercaptoundecanoate (1 and 2 in Fig. 2) 58 exhibited solubility of 0.4 and 3.4 mg/mL, respectively.Evaluated according to the threshold of drug concentration, the former is too low but the latter is high enough for the usage of intravenous injection.

Quantitative and site-directed modi¯cation to optimize the solubility and PDT activity
Previously, taurine substituted HB (3, Fig. 3) was synthesized, its aqueous solubility was more than 9 mg/mL, and the PDT activity was higher than sulfonate substituted HB but far lower than HB, 59 which suggested that prolonging the alkyl line in the substituent may lead to ¯nding the optimized or quantitative amphiphilicity.Generally, PDT activity of a photosensitizer is mainly dependent on two factorsthe cellular uptake and singlet oxygen yield. 603 exhibiting far lower PDT activity than HB is ascribed to lower cellular uptake and the lower singlet oxygen yield.An advantage of hypocrellins is that their structures are easily modi¯able by introduction of a substituent to site 2, 5, 8, 11, 13, 14 or 17, but the singlet oxygen yields of these derivatives are very di®erent.][65] According to these considerations, a series of 17amino-alkyl-sulfonic acid substituted derivatives of HB with 3, 4, 5 and 6 carbon atoms in the alkyl chain were synthesized (4-7 shown in Fig. 4). 35,64nterestingly, it was found that the aqueous solubility and PC formed linearly decreasing and increasing sequence with increase in the carbon number, in addition, the singlet oxygen yield increased gradually with the carbon atom number, as shown in Fig. 5.Among these derivatives, 6 exhibited the solubility of 1.7 mg/mL, which was just higher than the threshold of required drug concentration, and the singlet oxygen yield as high as 0.98.Consequently, it exhibited much higher PDT activity to human gastric cancer BGC823 cells than the parent HB, with the IC 50 (de¯ned as the photosensitizer concentration required killing 50% of the cells) of 22 nM compared with that of 40 nM for HB, in addition, it had no dark cellular toxicity. 35It can be concluded that 6 possesses the optimized amphiphilicity and PDT activity, and can be directly used for intravenous injection without need of preparation of drug-delivery vehicles.
Similarly, 15-deacetyl-13-amino-alkyl-sulfonic acid substituted HB derivatives (8 and 9, Fig. 6(a)) were designed and synthesized specially for PDT of AMD. 66These derivatives with the substituent at the site 13 exhibit the maximum absorption on orange light (580 nm), which may be a proper phototherapeutic window of AMD, because the light not only penetrates tissues no deeper than 1 mm coincided with the diseased targets, but also is less absorbed by visual pigments.Estimated similarly as mentioned above, the solubility of 7.1 or 2.0 mg/mL for 8 or 9 is large enough for intravenous injection, but the latter is better for e®ective cellular uptake.In vivo experiments prove that 9 results in much e®ective damage to blood vessels than 8 or HB, as shown in Fig. 6

Recognition of Biological Molecules by Environment-Sensitivity of Hypocrellin Fluorescence
In comparison to other kinds of photosensitizers, hypocrellin °uorescence is very sensitive to the microenvironment because it is originated from the intramolecular H-atom transfer process, 13 which means, any microenvironment to strengthen or weaken the intramolecular hydrogen bond will certainly a®ect the °uorescence.Imaginably, the environment-sensitive °uorescence could be taken as a probe to monitor the microenvironments of biological molecules or tissues.As mentioned above, joint usage of PDD and PDT for treatment of tumors at an early stage may be a right strategy to cure cancers, however, ¯nding a speci¯c di®erentiation of a tumor cell from a normal one is still a great challenge.For a long time, it was known that tumor cells or tissues possess the characteristics of a large amount of lipid and neonatal collagen, but lower pH values, as well as over expression of biological molecules, [67][68][69] however, the quantities all being relatively speci¯c but not absolutely speci¯c, make °uorescence diagnosis di±cult.According to the quantitative di®erence of the tumor cells from normal ones, it may be asked whether environmentsensitive °uorescence of hypocrellins can be used to recognize di®erent microenvironments, which may lead to some new clues for early diagnosis of tumors.

°uorescence of hypocrellins
Previously, it was reported that both bulk e®ect and polarity of solvents had a very pronounced impact on hypocrellin °uorescence, which was ascribed to the particular intramolecular hydrogen bond or intramolecular proton transfer between the keto and enol groups in a hypocrellin molecule. 13,73It was further con¯rmed by the methylation of HB eliminating the °uorescence completely. 14In fact, the °uorescence intensity of porphyrin-like photosensitizers also varied in the solvents with various polarity, but it was completely due to the solvent dependent absorbance. 70Therefore, the particular °uorescence may be valuable for monitoring the microenvironments of biological molecules or targets.

Using hypocrellin °uorescence for recognition of various biological molecules
The biological molecules on cellular surface include three main kinds including proteins, polysaccharides and lipids.Previously, hypocrellin °uorescent responses to the microenvironments of various biological molecules were investigated and the results were summarized below.

Speci¯c binding of HB with human serum albumin (HSA)
HSA is one of the main drug transporters in human blood plasma and contains three homologous domains (labeled I-III) which are divided into two sub-domains (A and B).It was ever reported that hypocrellins could randomly bind to the hydrophobic positions of HSA. 71Taking HB and the sole tryptophan in sub-domain IIA of HSA as dual °uorescence probes, it was clari¯ed that HB speci¯cally bound to the site I of HSA with the molar ratio of 1:1, suggested by the common in°ection point of the °uorescence intensity, at the concentration ratio of HSA and HB to 1:1, as shown in Fig. 7. 72,73 3.2.2.Speci¯c binding of HB to hyaluronan (HYA) HYA is a polysaccharide molecule playing an important part in life process, and over-expression in tumor cells or tissues. 74Previously, the binding and interaction of HB with HYA was investigated by monitoring the spectral responses of HB. 75 Interestingly, with a continuous increase in the concentration of HYA, the absorbance of HB continuously rose until a saturated value, but the °uorescence decreased until quenching completely.Based on the particular °uorescent property of HB and one time increase in the particle size of HYA after interaction with HB, the absorbance increasing relative to that in PBS, was ascribed to binding with the hydrophobic area of HYA, while the °uorescent quenching was ascribed to formation of two intermolecular hydrogen bonds, which completely inhibited the intramolecular proton transfer, as shown in Fig. 8.It was found that both the absorption and °uorescence increased when HB was bound to sodium alginate (SOA), another polysaccharide molecule, 75 suggesting a speci¯c binding of HB to HYA.

Quantitative °uorescence recognizing various kinds of biological molecules and monitoring binding speci¯city
By the use of HSA, bovine serum albumin (BSA) and ovalbumin (OVA) as the models of proteins, HYA and SOA as the models of polysaccharides, and liposome as mimic cell membranes, the spectral responses of HB to the microenvironments in various biological molecules were investigated. 76According to the absorption and °uorescence spectra, a parameter of R F=A , de¯ned as the ratio of the °uorescence to absorbance of HB, was proposed to characterize a microenvironment speci¯cally.Dependence of R F=A values of HB (8 M) on the normalized biomolecular concentrations or water, benzene solution were shown in Fig. 9. Generally, the R F=A values of HB form a decreasing sequence in benzene>liposome>protein>water>polysaccharide, which is similar to the sequence of decreasing in the hydrophobicity.For HB in benzene has a perfect hydrophobic environment, it has the highest R F=A value.HB in liposome is completely enclosed by the bilayer phospholipid membranes, but the R F=A value is far lower than in benzene, suggesting that the environment in liposome is not perfectly hydrophobic for presence of some water molecules in semi-°uid liposome. 77In comparison, the hydrophobic area in the proteins is not completely closed as in liposome, therefore the R F=A values are lower than in liposome.Particularly, hydrophobic area in polysaccharides is half-open, therefore, it has the lowest R F=A value.Although the microenvironment in polysaccharides is more hydrophobic than in water, which should result in increasing of both the absorbance and °uorescence, the R F=A values even lower than in PBS is ascribed to a less increase in °uorescence than in absorbance.The R F=A value of HB in HYA is far lower than that in SOA, due to the speci¯c binding of the former but non-speci¯c binding of the latter.Similarly, as a result of HB speci¯cally binding with HSA, the R F=A value of HB in HSA is far higher than that in BSA and OVA.
The R F=A values in BSA and OVA are almost identical, indicating a similar microenvironment.
Compared to other kinds of photosensitizers whose °uorescence is quantitatively proportional to the absorbance, hypocrellins exhibit very distinct °uorescent feature, which may be quantitatively used to recognize various biological microenvironments.It is well known that tumor and normal cells have quantitative di®erences in expression of some biological molecules, 11,12,78 therefore, recognizing various biological molecules by the quantitative spectral parameters of HB may provide a clue for quantitatively distinguishing the microenvironments of tumor cells from normal ones.

pH-sensitive °uorescence of hypocrellins
Compared to the normal tissues, tumor tissues exhibit lower pH values. 79Previously, °uorescent responses of hypocrellins to pH values were investigated.Figure 10 showed the °uorescent spectra The °uorescence spectra of HB in PBS or in liposome were also monitored in a series of pH values from 6.0 to 8.0. 80Interestingly, the °uorescence of HB in PBS exhibited the minimum at pH 7.0 while the maximum at pH 7.4 in liposome, as shown in Fig. 11, indicating that HB °uorescence is quantitatively sensitive to pH values in various microenvironments.

Conclusions and Perspectives
Generally, the drug-delivery problem for hypocrellins was solved by two strategiespreparing drugdelivery vehicles and ¯nding quantitatively amphiphilic derivatives.Among the drug-delivery vehicles, liposomal powder formulation is most practical, for not only its PDT activity and stability, but also facile manufacture on a large scale.On the other hand, nanoparticles of HB with proteins or polysaccharides are stable and PDT e®ective, but controlled-release drugs make it not suitable for PDT of microvascular diseases.The nanoemulsion of hypocrellins is the most PDT e®ective, but its low stability does not satisfy the pharmaceutical standards.Commonly, all of the drug-delivery vehicles exhibit lower PDT activity than the parents, HA or HB.As a new strategy, ¯nding the derivatives with quantitative amphiphilicity and optimized photosensitization activity, not only solves the drug-delivery problem of hypocrellin photosensitizers, but also achieves higher PDT activity than the parents.In consideration of the spectral properties of hypocrellins and characters of the diseased targets, it can be concluded that the amphiphilic hypocrellin derivatives may be suitable for PDT of not only some microvascular diseases, but also other kinds of super¯cial diseases.Joint usage of PDD and PDT to treat tumors at an early stage may be a correct strategy to cure various tumors ¯nally, however, to speci¯cally di®erentiate a tumor cell or tissue from normal one by PDD is still a great challenge, because of lack of not only a speci¯c marker from tumors, but also speci¯c °uorescence of a photosensitizer.It is well known that over-expression of some biological molecules in tumor cells or tissues are a character of tumors, compared to normal cells or tissues.However, these are quantitatively but not qualitatively speci¯c, that is, these molecules are also present in normal cells or tissues.Since presence of the quantitative di®erences between tumor and normal cells or tissues, logically, it may be asked whether the quantitative °uorescence responses can be used to recognize tumors.Particularly, hypocrellin °uorescence is environment-sensitive due to the intramolecular proton transfer mechanism, which is very distinct from other kinds of photosensitizers.As mentioned above, a speci¯c spectral parameter of hypocrellins could not only recognize various kinds of biological molecules, but also identify a speci¯c binding or nonspeci¯c binding with same kind of biological molecules.These may provide a possibility to quantitatively di®erentiate the microenvironments of tumor from normal cells or tissues, however, further studies are certainly necessary to achieve the ¯nal goal.

Fig. 1 .
Fig.1.The chemical structures of HA and HB.

Fig. 5 . 5 J
Fig. 5. (a) Plot of the PC or the solubility of 3-7 to the carbon atom number in the substituent.(b) Plot of the singlet oxygen quantum yield of 3-7 to the carbon atom number in the substituent.

Fig. 6 .
Fig. 6.(a) The molecular structures of 8 and 9. (b) PDT e®ects of HB (B), 8 (C) or 9 (D) on the blood vessels of CAM along with that of without photosensitizer as a control (A).First or second line is the blood vessels images of CAM recorded before irradiation or 6 h after PDT.

Fig. 7 . 7 J
Fig. 7.The plots of the °uorescence intensity of the tryptophan at 337 nm (a) and HB °uorescence at 620 nm (b) to the ratio of concentration of HB to HSA (10 M) in PBS (pH 7.4).(c) HSA structure and the speci¯c binding site.72

Fig. 9 .Fig. 8 .
Fig. 9. R F=A values of HB (8 M) in benzene solution, water and plot of R F=A values of HB (8 M) as a function of normalized concentrations of liposome, HSA, BSA, OVA, SOA and HYA in PBS (pH ¼ 7.0).