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Apr 29, 2016 - (Mppa), and make the photosensitizer potential in deep tumor treatment. The synthesis, spectral properties and in vitro photodynamic therapy ...
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A Novel Photosensitizer 31,131-phenylhydrazine -Mppa (BPHM) and Its in Vitro Photodynamic Therapy against HeLa Cells Wenting Li † , Guanghui Tan *, Jianjun Cheng † , Lishuang Zhao † , Zhiqiang Wang * and Yingxue Jin * Key Laboratory for Photonic and Electronic Bandgap Materials, Ministry of Education, College of Chemistry & Chemical Engineering, Harbin Normal University, Harbin 150025, China; [email protected] (W.L.); [email protected] (J.C.); [email protected] (L.Z.) * Correspondence: [email protected] (G.T.); [email protected] (Z.W.); [email protected] (Y.J.); Tel.: +86-451-8806-0569 (G.T. & Y.J.); +86-451-8806-0570 (Z.W.) † These authors contributed equally to this work. Academic Editors: Marino Resendiz and Scott Reed Received: 6 February 2016; Accepted: 23 April 2016; Published: 29 April 2016

Abstract: Photodynamic therapy (PDT) has attracted widespread attention due to its potential in the treatment of various cancers. Porphyrinic pyropheophorbide-a (PPa) has been shown to be a potent photosensitizer in PDT experiments. In this paper, a C-31 ,131 bisphenylhydrazone modified methyl pyropheophorbide-a (BPHM) was designed and synthesized with the consideration that phenylhydrazone structure may extend absorption wavelength of methyl pyro-pheophorbide-a (Mppa), and make the photosensitizer potential in deep tumor treatment. The synthesis, spectral properties and in vitro photodynamic therapy (PDT) against human HeLa cervical cancer cell line was studied. Methyl thiazolyl tetrazolium (MTT) assay showed the title compound could achieve strong inhibition of cervical cancer cell viability under visible light (675 nm, 25 J/cm2 ). Cell uptake experiments were performed on HeLa cells. Morphological changes were examined and analyzed by fluorescent inverted microscope. In addition, the mechanism of the photochemical processes of PDT was investigated, which showed that the formation of singlet oxygen after treatment with PDT played a moderate important role. Keywords: photodynamic therapy (PDT); BPHM; cell cytotoxicity

1. Introduction Cancer has been seriously threatening human health for a long period. Diagnosis and treatment of cancer has become the most pressing concern in contemporary society. Many therapeutic methods have been developed over the past decades, such as chemotherapy, radiotherapy and surgical therapy. Each of these approaches has disadvantages and drawbacks, such as incomplete treatment, side effects, patient suffering and high cost, and one should balance the disadvantages and the therapeutic effect. Among various choices for cancer treatment, photodynamic therapy (PDT) as a noninvasive therapeutic modality providing painless and repeating treatment for patients, has recently obtained regulatory approval for various cancers, including skin tumors [1], non-small-cell lung cancer [2], urinary system tumors [3], breast cancer [4], head and neck cancer [5], digestive system tumors [6]; hence, PDT has a good foreground and will provide alternative therapeutic methods. In PDT, the photosensitizer would attack any tissues it encounters. Because the photosensitizer may be excited by appropriate wavelength of light, it then passes on the excess energy to surrounding molecular oxygen, resulting in the generation of reactive oxygen species (ROS) [7]. These ROS are free radicals (Type I PDT) that generated through electron transfer from a substrate molecule, or Molecules 2016, 21, 558; doi:10.3390/molecules21050558

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highly reactive state of oxygen known as singlet oxygen (Type II PDT). Photosensitizer is a critical factor in PDT. An ideal photosensitizer should not only have high efficiency in generating ROS within the aerobic tissues but also have characteristics of low dark toxicity, easily targeted aggregation and long wavelength absorption (>600 nm) [8]. The earliest photosensitizers applied in clinic are hematoporphyrin derivative (HPD) and ‘photofrin’ [9,10], which has a number of shortcomings, such as complicated components, weak absorption band in the red region (630 nm), long-term cutaneous photosensitivity (about three months) and prolonged in vivo metabolism. Subsequently developed hematoporphyrin monomethyl ether (HMME) and 5-Aminolevulinic acid (5-ALA) in the 1990s were single component photosensitizers with better stability than HPD and ‘photofrin’ [11,12], yet they were not suitable for deep-seated tumors for the light being absorbed by the photosensitizers (667 nm), low dark toxicity, high molar extinction coefficient and high rate of fluorescence quantum yield [23]. C-3, 5, 7, 10, 12, 13, 17, 20 of MPPa are high-active reactive sites and are easily modified for the development of new photosensitizers. On the other hand, it is widely acknowledged that increasing the π-conjugation extending from the porphyrin core can lead to enhanced absorption properties [24]. In this present study, we made a modification on C-3, C-13 of Mppa by p-CF3 -phenylhydrazine to produce a new chlorin-based compound to develop photosensitizers in PDT. We consider that modification by p-CF3 -phenylhydrazin may increase the π-conjugation extending from the porphyrin core and shift absorption towards longer wavelengths. In general, the longer the wavelength, the deeper the penetration into tissues [25,26]. Red light can be used successfully for deeper localized targets; consequently, compounds with longer absorption wavelengths can be used in the treatment for deeper tissues. In this paper, the synthesis, the UV-visible spectroscopy and fluorescence spectroscopy of 1 C-3 ,131 bisphenylhydrazone modified methyl pyropheophorbide-a (BPHM) were studied. Moreover, in vitro photodynamic therapy (PDT) against the human HeLa cervical cancer cell line was studied by MTT assay to evaluate the title compound as the photosensitizer agent in support of PDT. Cell uptake experiments were performed in order to investigate the intracellular distribution. Morphological changes of HeLa cells after PDF treatment were analyzed by fluorescent inverted microscope. In addition, the photochemical processes mechanism of PDT was investigated by using specific quenching agent sodium azide (SA) and D-mannitol (DM) [27,28], respectively. 2. Results and Discussion 2.1. Chemistry The starting material Methyl pyropheophorbide-a (Mppa) was synthesized according to our previously reported procedure [29] (Scheme 1). Firstly, Mppa was hydrolyzed by hydrobromic acid in acetic acid, then oxidized by N(t-Pr)3 RuO4 to obtain C-31 ,131 -dicarbonyl product (3), which reacted with p-CF3 -phenylhydrazine under reflux conditions to obtain the end-product C-31 ,131 -Bisphenylhydrazone modified methyl pyropheophorbide-a (BPHM, 4). In practice, we initially performed the reaction under a moderately acidic condition at room temperature, yet it took a long time (>24 h) to complete the reaction. We assumed the conjugation between the carbonyl groups and cyclic π-aromatic system in the compound reduced the activity of nucleophilic addition. When compound 3 reacted with p-CF3 -phenylhydrazine under reflux, it only needed 5 h to obtain the target compound with a high yield.

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Scheme 1. The synthesis of BPHM (C-31,131-Bisphenylhydrazone).

1 1 1 -Bisphenylhydrazone). Scheme Scheme1.1.The Thesynthesis synthesisofofBPHM BPHM(C-3 (C-3,13 ,131-Bisphenylhydrazone).

2.2. Optical Properties

2.2. 2.2.Optical OpticalProperties Properties

Appropriate wavelength of light penetrates healthy tissue to reach the lesion, then activates the

1,131 of photosensitizer in focal tissues, leading to damage. Modifications by phenylhydrazone on C-3 Appropriate ofoflight healthy tissue activates the Appropriatewavelength wavelength lightpenetrates penetrates healthy tissueto toreach reachthe thelesion, lesion,then then activates the 1 1 1 Mppa lead to compounds with longer absorption wavelength, for it can effectively increase the photosensitizer phenylhydrazoneon onC-3 C-3,13 ,131of photosensitizerininfocal focaltissues, tissues,leading leadingto to damage. damage. Modifications by phenylhydrazone π-conjugation system extending from the porphyrin core, resulting in enhanced absorption properties. ofMppa Mppalead leadtotocompounds compounds with with longer longer absorption absorption wavelength, for itit can caneffectively effectivelyincrease increasethe the Hence, it is necessary to investigate the optical properties of BPHM. π-conjugation systemextending extendingfrom fromthe theporphyrin porphyrincore, core,resulting resultingininenhanced enhancedabsorption absorptionproperties. properties. π-conjugation system Figure 1a shows the ultraviolet-visible absorption of Mppa and BPHM recorded in MeOH Hence,ititisisnecessary necessarytotoinvestigate investigatethe theoptical opticalproperties propertiesofofBPHM. BPHM. Hence, (2.15 × 10−5 M), respectively. Both Mppa and BPHM demonstrate the characteristic adsorption of Figure 1a shows the ultraviolet-visible absorption of Mppaand andBPHM BPHMrecorded recordedininMeOH MeOH Figure 1a shows the ultraviolet-visible absorption of Mppa chlorins, which absorb throughout the ultraviolet region into the visible region between about 300 ´−5 5 M), (2.15and M), respectively. Both Mppa and BPHM demonstrate the characteristic adsorption (2.15 ˆ×1010 respectively. Both Mppa and BPHM demonstrate the characteristic adsorption 800 nm. Strong Soret absorption peaks appear in the range 350–420 nm. The Soret absorption peak ofof chlorins, which absorb throughout ultraviolet region the visible region about 300 chlorins, which absorb throughout thethe ultraviolet region the visible region between 300 and of BPHM increased obviously compared with Mppa, yetinto theinto absorption wavelength isbetween notabout changed. andnm. 800BPHM, nm. Strong absorption peaks appear instrongest the range 350–420 nm. The Soret absorption peak 800 Strong SoretQSoret absorption appear in the range 350–420 nm. The Soret absorption For four peaks appearpeaks in visible area. The absorption is Qy peak, which displayspeak a marked 16 nm obviously redshift and an obvious increase of peak intensity compared to Mppa. is The molar BPHM increased obviously compared with Mppa, yetthe theabsorption absorption wavelength isnot not changed. ofofBPHM increased compared with Mppa, yet wavelength changed. 4 L/mol·cm calculated based on Beer-Lambert Law. extinction coefficient is 5.88 × 10 ForBPHM, BPHM,four fourQQpeaks peaksappear appearininvisible visiblearea. area.The Thestrongest strongestabsorption absorptionisisQy Qypeak, peak,which whichdisplays displays For marked1616nm nmredshift redshiftand andananobvious obviousincrease increaseofofpeak peakintensity intensitycompared comparedtotoMppa. Mppa.The Themolar molar a amarked 4 4 extinctioncoefficient coefficientisis5.88 5.88ˆ×10 10 L/mol¨ L/mol·cm extinction cmcalculated calculated based based on on Beer-Lambert Beer-Lambert Law. Law.

Figure 1. Absorption (a) and fluorescence spectra (b) of Mppa, BPHM in CH3OH (2.15 × 10−5 M for UV absorption spectrum, 2.5 × 10−7 M for fluorescence spectrum).

´5−5M Figure1.1.Absorption Absorption(a)(a)and and fluorescence spectra Mppa, BPHM in CH 3OH (2.15 × 10 Mfor for Figure fluorescence spectra (b)(b) of of Mppa, BPHM in CH (2.15 ˆ 10 3 OH ´7 −7 UVabsorption absorptionspectrum, spectrum,2.5 2.5ˆ×10 10 M UV M for for fluorescence fluorescence spectrum).

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4 of 12 conjugated pyrrole rings, porphyrins possess high rigidness and better coplanarity, exhibiting strong fluorescence at room temperature. After being excited by light, the Because of the four conjugated pyrrole rings, porphyrins possess high rigidness and better emitted visible light from photosensitizers is very helpful in photodynamic clinical diagnosis. Figure 1b coplanarity, exhibiting strong fluorescence at room temperature. After being excited by light, the emitted shows the fluorescence spectrograms of Mppa and BPHM recorded in MeOH (2.5 ˆ 10´7 M), visible light from photosensitizers is very helpful in photodynamic clinical diagnosis. Figure 1b respectively. The obtained fluorescence spectra of Mppa and BPHM are both symmetric after being shows the fluorescence spectrograms of Mppa and BPHM recorded in MeOH (2.5 × 10−7 M), excited by 413 nmThe visible light,fluorescence and the emission are 670 nm nm, respectively. respectively. obtained spectrawavelength of Mppa and BPHM areand both685 symmetric after being

excited by 413 nm visible light, and the emission wavelength are 670 nm and 685 nm, respectively. 2.3. Photodynamic Activities 2.3. Photodynamic Activities After a photosensitizer is promoted to an excited state under light, it will produce singlet oxygen, which would lead to high cytotoxicity to target In thisstate part,under HeLalight, cells were to detect the After a photosensitizer is promoted to cells. an excited it willused produce singlet anticancer activity of our designed compound. The viability of HeLa cells incubated with different oxygen, which would lead to high cytotoxicity to target cells. In this part, HeLa cells were used to concentrations of BPHMactivity and Mppa were analyzed by MTT The assay. detect the anticancer of our designed compound. viability of HeLa cells incubated with Figure 2 concentrations showed the photodynamic of the title compound and Mppa against HeLa cells. different of BPHM and activity Mppa were analyzed by MTT assay. Black columns stand for the cell viability of BPHM experiment groups, red columns stand the Figure 2 showed the photodynamic activity of the title compound and Mppa against HeLafor cells. cell viability of Mppa experiment blue experiment columns for the cell viability control groups. Black columns stand for the cell groups, viability and of BPHM groups, red columnsofstand for the cell of Mppa(blue experiment groups, andwith blue different columns for the cell viability of control groups. The viability control groups columns) treated concentrations of BPHM (1, 5, 10, 20,The 30, control groups (blue columns) treated with different concentrations of BPHM (1, 5, 10, 20, 30, 40, 50, 60 40, 50, 60 and 120 µM) without light show high cell viability (>90%), which suggests that BPHM and 120 μM) withoutthe light showcell highwithout cell viability whichthe suggests hardly could hardly influence cancer light.(>90%), However, BPHMthat andBPHM Mppacould experiment influence cancer light. However, theofBPHM clearlyrate showed clearly showedthe that withcell the without increasing concentrations BPHMand andMppa Mppa,experiment the cell viability is on that with the increasing concentrations of BPHM and Mppa, the cell viability rate is on the decline, the decline, suggesting that both BPHM and Mppa have good cell growth inhibiting ability. When suggesting that both BPHM and Mppa the havecell good cell growth When the the concentration of BPHM reached to 50µM, viability lowed toinhibiting 10.99% ˘ability. 1.26%, suggesting concentration of BPHM reached to 50μM, the cell viability lowed to 10.99% ± 1.26%, suggesting that that the BPHM had strong photodynamic effects on the HeLa cells. Moreover, the cell viability of the BPHM had strong photodynamic effects on the HeLa cells. Moreover, the cell viability of Mppa Mppa was slightly higher than BPHM (p < 0.05, the difference was significant), and the IC50 values was slightly higher than BPHM (p < 0.05, the difference was significant), and the IC50 values of BPHM of BPHM and Mppa under visible light (675 nm,2 25 J/cm2 ) are 9.21 ˘ 0.91 µM and 12.90 ˘ 0.53 µM, and Mppa under visible light (675 nm, 25 J/cm ) are 9.21 ± 0.91 μM and 12.90 ± 0.53 μM, respectively. respectively. Although our designed compound did not show an obvious advantage compared to Although our designed compound did not show an obvious advantage compared to Mppa, it has Mppa, it has longer absorption wavelength than that of Mppa, which gives it more potential in deep longer absorption wavelength than that of Mppa, which gives it more potential in deep tumor tumor treatment. In addition, to the phenylhydrazine our compound possesses relatively treatment. In addition, duedue to the phenylhydrazine part,part, our compound possesses relatively high highlipid lipidsolubility, solubility,which whichmakes makes it easier to permeate cell membranes and enter the cells. All in all, it easier to permeate cell membranes and enter the cells. All in all, our our designed compound has long absorption wavelength and slightly higher cell toxicity than Mppa. designed compound has long absorption wavelength and slightly higher cell toxicity than Mppa. BPHM could kill kill the the cellcell effective under thethe light and feasibilityin in BPHM could effective under light andthe thelow lowdark darktoxicity toxicityprovides provides the the feasibility clinical application. clinical application.

Figure 2. Cell viability of three groups: BPHM experimentgroups groups(black (blackcolumns), columns), Mppa Mppa experiment Figure 2. Cell viability of three groups: BPHM experiment experiment groups (red columns) and control groups (blue columns). Each group was cultured with different groups (red columns) and control groups (blue columns). Each group was cultured with different concentrations of BPHM or Mppa 5, 10, 50,6060and and120 120µM, μM,200 200 µL), μL), cell cell viability viability was concentrations of BPHM or Mppa (1,(1, 5, 10, 20,20, 30,30, 40,40,50, was assessed by MTT after 24 h. Statistically significant between and experiment Mppa experiment assessed by MTT assayassay after 24 h. Statistically significant between BPHMBPHM and Mppa groups were by performed by0.05 t-test, p < 0.05 showed the difference was significant). weregroups performed t-test, p < showed the difference was significant).

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In order to investigate the mechanism of photochemical processes (Type I and Type II) in PDT, 2.4. Formation Reactive SpeciesIIingenerated PDT corresponding ROSof of TypeOxygen I and Type from photodynamic reaction were quenched by using specific agent sodium azide (SA) and Dprocesses -mannitol (DM), respectively [27,28]. In orderquenching to investigate the mechanism of photochemical (Type I and Type II) in PDT, corresponding ROSD-mannitol of Type I and Typecould II generated from react photodynamic reaction quenched by Sodium azide (SA) and (DM) selectively with oxygen freewere radicals and singlet quenchingmaking agent sodium azide (SA) and DROS -mannitol (DM), respectively [27,28]. oxygenusing (1 O2specific ), respectively, the corresponding inoperative on cancer cells.Sodium Figure 3 azide and D-mannitol could PDT selectively react with oxygen on freecytotoxicity radicals and singlet showed the(SA) influences of three(DM) different processing methods effects.oxygen The cell (1O2), respectively, making the corresponding ROS inoperative on cancer cells. Figure 3 showed the viability of BPHM-PDT-SA group and BPHM-PDT-DM group was a little higher than that of the influences of three different PDT processing methods on cytotoxicity effects. The cell viability of BPHM-PDT group, suggesting that the key ROS have been quenched, resulting in low cytotoxicity BPHM-PDT-SA group and BPHM-PDT-DM group was a little higher than that of the BPHM-PDT to HeLa cells.suggesting Moreover, the BPHM-PDT-SA groupinwas higher thancells. that of group, that thecell keyviability ROS haveofbeen quenched, resulting lowslightly cytotoxicity to HeLa BPHM-PDT-DM group at various concentrations (1–120 µM) of BPHM, demonstrating that in Moreover, the cell viability of BPHM-PDT-SA group was slightly higher than that of BPHM-PDT-DMvitro Type I group and Type II photodynamic reactions occurred simultaneously, yetthat Type reaction at various concentrations (1–120 μM) of BPHM, demonstrating in IIvitro Type played I and a moderate role. reactions occurred simultaneously, yet Type II reaction played a moderate Typeimportant II photodynamic role.earlier, in the Type I photodynamic reaction, photosensitizer could interact with Asimportant mentioned As mentioned earlier, in the Type I photodynamic photosensitizer interactperoxide with any active substrate, producing hydroxyl radicals (HR),reaction, superoxide anion, orcould hydrogen any active substrate, producing hydroxyl radicals (HR), superoxide anion, or hydrogen peroxide by by electron transfer process. We inferred that the cancer cells had provided some integral substrate electron transfer process. We inferred that the cancer cells had provided some integral substrate for for photosensitizer to complete the Type I reaction during the in vitro test. For Type II reaction, the photosensitizer to complete the Type I reaction during the in vitro test. For Type II reaction, the excitation energy might be transferred to nearby tissue oxygen, producing singlet oxygen, that is, the excitation energy might be transferred to nearby tissue oxygen, producing singlet oxygen, that is, the Type IIType reaction would take take place generally in in thethe presence oxygen,yet yetininour our experiments, II reaction would place generally presenceof oftissue tissue oxygen, cellcell experiments, the excitation energy might be transferred to to cellular Therefore,coexistence coexistence of Type I and the excitation energy might be transferred cellularcomponents. components. Therefore, of Type I and Type IIType was IIreasonable in practice. was reasonable in practice.

Cell viability of BPHM-PDT, BPHM-PDT-SA,and andBPHM-PDT-DM BPHM-PDT-DM group PDT treatment FigureFigure 3. Cell3.viability of BPHM-PDT, BPHM-PDT-SA, groupafter after PDT treatment 24 h, respectively. BPHM-PDT (black column) stands for the groups treated with BPHM under light 24 h, respectively. BPHM-PDT (black column) stands for the groups treated with BPHM under light (675 nm). BPHM-PDT-SA (red column) stands for the groups treated with BPHM and Sodium azide (675 nm). BPHM-PDT-SA (red column) stands for the groups treated with BPHM and Sodium azide under light (675 nm). BPHM-PDT-DM (blue column) stands for the groups treated with BPHM and under light (675 nm). BPHM-PDT-DM (blue column) stands for the groups treated with BPHM and D-mannitol under light (675 nm). All of the groups were cultured with different concentrations of BPHM D -mannitol under light (675 nm). All of the groups were cultured with different concentrations of (1, 5, 10, 20, 30, 40, 50, 60 and 120 μM, 200 μL), the concentration of SA was 20 mmol/L, DM was BPHM40 (1,mmol/L 5, 10, 20, 30,viability 40, 50, 60 and 120 µM, 200 µL), thethiazolyl concentration of SA was 20 mmol/L, Cell was determined by methyl tetrazolium (MTT) assay. A t-testDM waswas 40 mmol/L Cell viability was determined by methyl thiazolyl tetrazolium (MTT) assay. A t-test performed according to the IC50 of each group. p < 0.05 (t-test) between BPHM and BPHM-PDT-SAwas performed according to the IC50 of group.was p < significant; 0.05 (t-test)p between BPHM and BPHM-PDT-SA experiment groups showed theeach difference < 0.05 (t-test) between BPHM and experiment groups showed the groups difference wasthesignificant; p