Encapsulation efficacy of natural and synthetic photosensitizers by ...

IET Nanobiotechnology Research Article

Encapsulation efficacy of natural and synthetic photosensitizers by silica nanoparticles for photodynamic applications

ISSN 1751-8741 Received on 6th January 2015 Revised on 26th May 2015 Accepted on 3rd June 2015 doi: 10.1049/iet-nbt.2015.0003 www.ietdl.org

Ghaseb Naser Makhadmeh 1,2 ✉, Azlan Abdul Aziz 1, 2, Khairunisak Abdul Razak 2, 3, Osama Abu Noqta 1, 2 1

School of Physics, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia NanoBiotechnology Research and Innovation (NanoBRI), Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800 USM, Penang, Malaysia 3 School of Materials and Mineral Resources Engineering, Universiti Sains Malaysia, 14300 Nibong Tebal, Penang, Malaysia ✉ E-mail: [email protected] 2

Abstract: This study analysed the physical effects of Cichorium Pumilum (CP), as a natural photosensitizer (PS), and Protoporphyrin IX (PpIX), as a synthetic PS, encapsulated with silica nanoparticles (SiNPs) in photodynamic therapy. The optimum concentrations of CP and PpIX, needed to destroy Red Blood Cells (RBC), were determined and the efficacy of encapsulated CP and PpIX were compared with naked CP and PpIX was verified. The results confirmed the applicability of CP and PpIX encapsulated in SiNPs on RBCs, and established a relationship between the encapsulated CP and PpIX concentration and the time required to rupture 50% of the RBCs (t50). The CP and PpIX encapsulated in SiNPs exhibited higher efficacy compared with that of naked CP and PpIX, respectively, and CP had less efficacy compared with PpIX.

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Introduction

Photodynamic therapy (PDT) is a new cancer treatment that utilises certain drugs that react with light. Exposing these drugs to light initiates a series of chemical reactions to produce singlet oxygen which leads to the destruction of cancer cells. However, the efficacy of 1O2 production decreases considerably because only a small amount of photosensitizer (PS) eventually reaches the intended target. The reduction of PS is attributable to the modification of the defence mechanism by reticulo-endothelial system or macrophage system which engulfs some of the PS in the body. Nevertheless, the effect of macrophage can generally be evaded by structures that are biocompatible and smaller than 100 nm such as silica and gold nanoparticles. Based on this phenomenon, the efficacy issue associated with interactions with macrophage can be resolved using nanoparticle as a carrier for PS. The high encapsulation quality of silica nanoparticles (SiNPs) is due to its intrinsic properties. SiNPs exhibit high biocompatibility, low toxicity, amorphous and transparent for the light which makes it an ideal material for drug delivery system rather than other types of nanoparticles such as gold nanoparticles that are not transparent to light for PS activation [1]. Other intrinsic features of SiNPs include: the ability to be synthesised without difficulty at low temperatures with low polydispersity, the ability to provide suitable sites for bio-molecular compounds to adhere to its outer surface, and the ability of its inner surface to encapsulate the PS [2–4]. Cichorium Pumilum (CP), as a natural and herbal PS [5] in lieu of the trade, has been approved as a drug. Herbal PSs absorb visible and near-ultraviolet light present in many organisms including fungi, higher plants, bacteria, protozoa, invertebrates and vertebrates. The sources of PSs in CP are the flowers and aerial parts and in this report we used their extract. Cichoriin, which is known to be sensitive to light, was extracted from CP’s flowers [6]. CP’s roots which were the source of Lactucin is also found to be sensitive to light [7, 8]. In this study, CP encapsulated SiNPs applicability in PDT was demonstrated in vitro study using Red Blood Cells (RBCs) because the RBCs are the vertebrate organism’s principal means of delivering oxygen (O2) to the body tissues via the blood

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flow through the circulatory system and also a key player in getting waste carbon dioxide from tissues to the lungs. Comparative analysis was performed between the efficacies of naked CP and encapsulated CP on RBCs by studying their effectiveness at different concentrations and exposure times. Protoporphyrin IX (PpIX) is derived from porphyrin [9–11]. The effect of PpIX starts with light energy absorption, which initiates a sequence of chemical reactions to generate singlet oxygen. The subsequent reaction of singlet oxygen with DNA is the phenomenon responsible for cancer cell destruction [12–14]. PpIX encapsulated by SiNP was examined in vitro to determine its effect on cancer cells. Measured parameters of PpIX-SiNPs include optimum concentration, optimal exposure and cytotoxicity. The efficacy of encapsulated PpIX was analysed against that of naked PpIX after establishing the most effective concentration of PpIX for different durations of exposure.

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Materials and methods

Five different concentrations of CP (0.364, 0.182, 0.091, 0.046 and 0.023 mg/ml as final concentration) were encapsulated with SiNPs. The encapsulation of CP by SiNPs was synthesised using reverse-micellar method [15]. The sample preparation procedure involves several steps. Firstly, RBCs were diluted in phosphate buffer saline to obtain the maximum concentration measurable at maximum absorption. The maximum peak of RBCs was near 2.0 at 577 nm. To test the applicability of encapsulated CP in SiNPs for PDT, the diluted RBCs were mixed with the five different concentrations of CP-SiNPs and exposed to the light source at different times (0, 15, 30, 45, 60, 75, 90, 105 and 120 min). The efficacy of Naked CP was also tested using five different concentrations of CP (0.364, 0.182 and 0.091 mg/ml as a final concentration) under light exposure of different times. Comparative analysis was performed between results for encapsulated CP-SiNPs and naked CP. The changes in the absorption spectra of all samples were studied. The relationship between the PS concentration and the time needed to destroy 50% of the RBCs (t50) (50% mortality) was also analysed using the

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method described by Al-Akhras et al. [16]. The dependence of delayed photohemolysis on the PS concentration is then expressed empirically by the underlying equations 1/t50 = k.C (p) Ln 1/t50 = Ln(k) + pLn(C)

(1) (2)

where k is constant, C is the PS concentration, and p is the power dependent parameter. For PpIX, different concentrations of encapsulated and naked PpIX were used to prove its photodynamic effect on RBCs. Five different concentrations of encapsulated PpIX (1.545, 0.773, 0.386, 0.193 and 0.097 µM as a final concentration) were used to damage the RBCs. Naked PpIX was also tested by using approximately 2.67 fold of encapsulated PpIX concentrations (4.156, 2.078, 1.039, 0.520 and 0.260 µM as a final concentration) and compared with the encapsulated PpIX results.

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Results

3.1 Fractional haemolysis and 50% mortality of RBCs by CP The size of encapsulated SiNPs, determined by TEM, was approximately 8 nm while the effect of hyperthermia on RBCs from the light source, used in this work, was shown to be insignificant [17, 18]. The toxicity of blank SiNPs was measured by incubating the maximum concentration of SiNPs (1 ml) in RBCs (4 ml) for different incubation times (0, 30, 60, 90, 120, 150 and 180 min). The cell survival was determined after that. The results (in Figs. 1a and b) show that the blank SiNPs kill 50% of the cells after 108 min which means that if the treatment finished before that time, then the treatment efficacy is mostly due to PDT and not by the blank SiNPs toxicity. The high cytotoxicity at t > 108 min must likely due to be the residual chemicals such as 1-butanol and surfactant used in SiNPs synthesis. Five different concentrations of encapsulated CP (0.364, 0.182, 0.091, 0.046 and 0.023 mg/ml as a final concentration) were used to destroy the RBCs, compared with three different concentrations of naked CP (0.364, 0.182 and 0.091 mg/ml). The maximum peaks at 577 nm for all absorption spectrums of different naked and encapsulated CP concentration decreased after prolonged exposure time as shown in Figs. 2a and b. The normalisation of the fitting curve was drawn to calculate the 50% mortality (t50) of RBCs for every concentration, as shown in Figs. 3a and b. This normalisation can be used to determine the 50% mortality of the RBCs during the treatment by the naked and encapsulated CP with exposure to the light. The results show that the 50% mortality of the RBCs increased with the decrease in CP concentration for naked and encapsulated CP, because the amount of produced singlet oxygen also increased and thus have faster time to destroy half of RBCs. RBCs were destroyed faster when treated by encapsulated CP, because the encapsulated CP molecules are clustered together by the SiNPs membrane, absorbed high energy light compared with the naked CP molecules that were spread everywhere in the sample. Fig. 4 has shown the relation between naked and encapsulated CP concentrations versus the 50% mortality of RBCs. The equation that related the encapsulated CP concentration and t50 was Ln 1/t50 = −2.7348 + 0.53558Ln(C)

(3)

The equation that related the naked CP concentration and t50 was: Ln 1/t50 = −2.4386 + 1.06730Ln(C)

(4)

The equation that related the t50 and CP concentration can be useful

Fig. 1 Cytotoxicity of SiNPs on RBCs at Maximum Concentration a Change in the maximum peak b Normalisation curve to measure the 50% mortality of RBCs

in estimating the optimum concentration or exposure time needed for constructing a good design of experiments (DOS) exercise. 3.2 Fractional haemolysis and 50% mortality of RBCs by PpIX For PpIX, different concentrations of encapsulated and naked PpIX were used to prove its photodynamic effect on RBCs. Five different concentrations of encapsulated PpIX (1.545, 0.773, 0.386, 0.193 and 0.097 µM as a final concentration) were used to damage the RBCs. Naked PpIX was also tested by using 2.67 fold of encapsulated PpIX concentrations (4.156, 2.078, 1.039, 0.520 and 0.260 µM as a final concentration) and compared with the encapsulated PpIX results. The decreasing of the maximum absorption peaks for all different concentrations of naked and encapsulated PpIX after enough exposure time can be shown in Figs. 5a and b; after PpIX absorbed enough light energy, all PpIX molecules produced 1O2 that was the main component capable of destroying the RBCs. The normalisation of the fitting curve was used to calculate the t50 of RBCs as shown in Figs. 6a and b. The t50 of the RBCs increased when the naked and encapsulated PpIX concentration was decreased. The reason for that is when the concentration increased, the amount of singlet oxygen that was produced also increased; thus, half of the RBCs were destroyed faster. RBCs were destroyed faster when they were treated by encapsulated PpIX because the encapsulated PpIX molecules

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Fig. 3 Normalisation of maximum peak for all samples Fig. 2 Reduction of the maximum peak for all samples

a Encapsulated CP and b Naked CP

a Encapsulated CP b Naked CP

absorbed high energy light compared with the naked PpIX molecules that spread in the sample. Fig. 7 has shown the relation between the PpIX concentration and the t50. The equation that related the encapsulated PpIX concentration and t50 was Ln 1/t50 c = −3.6558 + 0.23173Ln(C)

(5)

The reason of the changes in the spectrums, as shown in Fig. 3, was the singlet oxygen that was produced after exposing CP to the light source. The naked and encapsulated CP did not destroy the RBCs directly after exposure time because they need to absorb sufficient energy from the light to produce the singlet oxygen. Singlet oxygen damaged the membrane of RBCs thus the haemoglobin was spread in the outer liquid that caused the reduction in the RBCs absorption values. It is apparent from the results that RBCs were destroyed at less exposure time as CP concentration increased.

The equation that related the naked PpIX concentration and t50 was Ln 1/t50 = −4.2653 + 0.41619Ln(C)

(6)

Again, the equation that related the t50 and PpIX concentration can be useful in estimating the optimum concentration or exposure time needed for constructing a good DOS exercise.

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Discussion

The observed decrease in the RBCs spectra in Figs. 2 and 3 is attributable to the singlet oxygen produced following the exposure of CP to the light source. The singlet oxygen ruptured the RBCs membrane, resulting in the spread of haemoglobin in the sample, thus decreasing the absorption spectrums. The efficacy of naked CP at the same concentration was then compared with that of encapsulated CP. It is evident from the results that CP encapsulated by SiNPs have relatively higher efficacy in destroying RBCs compared with using naked CP because the CP molecules clustered together by the SiNPs encapsulation, causes high absorbance of light energy when in contact with RBCs.

Fig. 4 Relation between the 50% mortality of RBCs and the concentration of encapsulated and naked CP



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Fig. 5 Reduction of the maximum peak for all samples

Fig. 6 Normalisation of maximum peak for all samples

a Encapsulated PpIX b Naked PpIX

a Encapsulated PpIX and b Naked PpIX

When the concentration of CP was increased, the amount of produced singlet oxygen also increased resulting in higher rate at destroying half of RBCs as shown in Fig. 3. RBCs were destroyed faster when they were treated by encapsulated CP because more CP molecules are available to absorb high energy light compared with naked CP molecules that are spread everywhere in the sample. From the results, it can be deduced that SiNPs encapsulation allowed light to effectively penetrate through their membrane to reach the CP, which subsequently increases singlet oxygen production and the destruction of RBCs. This phenomenon is attributable to the fact that SiNPs encapsulation is able to secure clusters of CP molecules that subsequently increase the absorbance of light energy. However, for naked CP, the molecules are distributed randomly in the solution, and their interactions with light are slower and dispersed, resulting in low absorbance. The relation derived between t50 and concentration of naked CP and encapsulated CP can be applied in future works to calculate the most appropriate exposure time for optimal concentration without performing any experiments. We can conclude from the relation between the fractional haemolysis of RBCs and the exposure time during application of PDT on the RBCs that the encapsulated CP has higher efficacy than naked CP for concentration and exposure time. For concentration efficacy, encapsulated CP has higher efficacy than naked CP approximately by 46%. Moreover, for exposure time efficacy, encapsulated CP has higher efficacy than naked CP approximately by 98%. The decrease in the absorption spectrums of the PpIX trend started at the moment PpIX absorbed enough energy from the light source to produce the singlet oxygen that destroyed the RBCs as shown in

Figs. 5 and 6. The changes in the spectrums are caused by the singlet oxygen that was produced after exposing PpIX to the light source. Singlet oxygen destroyed the membrane of RBCs and as a result, the haemoglobin was spread into the fluid that caused a reduction in the absorption values. The encapsulated PpIX destroyed the RBCs faster than naked PpIX and required concentration less than that required by naked PpIX to have the

Fig. 7 Relation between the 50% mortality of RBCs and the concentration of encapsulated and naked PpIX


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same efficacy. It is obvious that the encapsulated PpIX have higher efficacy compared with the naked PpIX. The reasons for the higher efficacy of encapsulated PSs are that the PpIX molecules collected together by SiNPs and SiNPs are applicable with RBCs; thus, cause high absorbance of energy light and an increase in 1O2 production. However, compared with the naked PpIX, the PpIX molecules were spread evenly in the solution and its interaction with the light was slow and dispersed; thus, 1O2 production was less than the amount that was produced by encapsulated PpIX. The encapsulated PpIX has higher efficacy than naked PpIX for concentration and exposure time. For concentration efficacy, encapsulated PpIX has higher efficacy than naked PpIX approximately by 78%. Furthermore, for exposure time efficacy, encapsulated PpIX has a higher efficacy than naked PpIX approximately by 48%. The comparison between the efficacy of the concentration and exposure time for the natural and synthetic PSs (CP and PpIX) was studied. For the concentration efficacy, the PpIX has higher efficacy compared with the CP by 99% due to the high light intensity, because that depends on the maximum absorption peak, or/and the PpIX optical properties as PSs and the CP as a natural PS needs more extract to produce more efficacy. For the exposure time efficacy, the PpIX efficacy was higher and then the CP. The same reason for the concentration efficacy is due to the light intensity or/and the PSs optical properties. We can conclude that the natural PS, CP, had less efficacy comparing with the synthetic PS, PpIX and the SiNPs, as a drug delivery agent for PDT, had high bio-compatibility with the RBCs that were tested in this work to be used in PDT.

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Acknowledgment

This work was funded by USM RU grant (1001/PFIZIK/846087) and (1001/PFIZIK/814113).

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