Cationic Quaternized Chitosan Nanoparticles for C-phycocyanin Delivery to Hela Cells

In this study we synthesize C-PC/QAC nanoparticles using cationic quaternary ammonium chitosan (QAC) as substrate material loaded C-phycocyanin (C-PC) by ionic-gelation method. The prepared NPs was about 120nm size with spherical morphology, the entrapment efficient was 62%, and the drug-loading amount was 20%. The NPs exhibited a pH sensitive realease of C-PC in vitro. The drug-NPs were proved to be hemocompatible and safe. In conclusion, QAC increased bioavailability and anticancer property of C-PC.


INTRODUCTION
Nano-drug is a hot-spot in the field of medicine, which can overcome the limitations of conventional drug delivery systems [1]. With the development of nanoparticle technology, the study of nanoparticle as drug carrier has attracted broad attention. Cervical cancer is one of the first discovery of cancer and seriously threatening life and health among females worldwide [2]. C-phycocyanin (C-PC) is an important natural photosynthetic pigment with many advantages such as safety, non-toxicity, high hydrophilcity and good dyeing force. Its absorption peak at 620nm is strongest [3,4]. Li bing's studies found that C-PC has anti-tumor and immune-enhancing effect [5]. However, its application in medical field is limited by the poor stability. Therefore, nano-drugs provide a good choice to set up an efficient delivery for C-PC. Quaternary ammonium chitosan (QAC), a kind of chitosan derivative, had an attention in drug delivery application [6]. It can load C-PC to form nanoparticles and achieve slow-release effect.
Human cervical cancer cells (Hela) and fresh human blood were provided by the Affiliated Hospital of the

Preparation of C-PC/QAC-NPs
The C-PC/QAC-NPs were made by the simple ionic-gelation with crosslinking agent tripolyphosphate (TPP) [7,8] (1) and (2)) (2) Wnps where C1 is the total C-PC concentration in the NPs solution, C2 is the C-PC concentration in the supernatant, V is the total volume of prepared NPs solution, and Wnps is the total amount of NPs.

Evaluation of nanoparticles
The particle size and surface morphology of NPs was analyzed using Scanning Electron Microscope (SEM/JEOL JSM-840 Analytical Scanning Electron Microscope).

In vitro drug release studies
In vitro C-PC release from C-PC/QAC-NPs was determined by dialysis method under two different pH5. 6 and 7.4 at 37 o C as described in literature. The prepared NPs suspension was centrifuged at 12000 rpm for 15 min and the collected pellets were redispersed in 5mL of PBS, then filled in a dialysis tubes, and put in a beaker containing 30 ml PBS (pH5. 4 and 7.4) at 37 o C with gentle stirring in a shaking incubator. The release studies were carried at fixed time intervals : 1, 2, 4, 8, 12, 24, 48h up

Hemolysis assay in vitro
Hemolytic activity of C-PC, QAC-NPs and C-PC/QAC-NPs was evaluated according to the previous protocol. Fresh blood from human volunteers was added into the tubes with acid citrate dextrose centrifugated in order to obtain the red blood cells (RBCs). After washing and diluting, the RBCs suspension were added to C-PC, C-PC/QAC-NPs and QAC-NPs solution. Then incubated at 37 o C for 2h.
PBS and Trixon X-100 (10g/L) were used as negative and positive controls, respectively. After incubation, the RBCs were centrifuged at 3000rpm for 10 min.
Free hemoglobins in the supernatant were measured spectrophoto-metrically using Beckman Coulter Elisa plate reader at 540nm. Hemolysis ratio (HR) of RBCs was calculated based on the equation [10].

Statistics analysis
The data were expressed as mean ± SD. A Student's t-test was used to determine the significance. Data were considered to be statistically significant at P˂0.05.
SPSS software was used for statistical analysis.

Synthesis of C-PC loaded QAC nanoparticles
C-PC/QAC-NPs were obtained as a result of chemical cross-linking with triphosphoric acid (TPP). The preincubation of C-PC with QAC was carried out to facilitate electrostatic interaction between the anionic C-PC and cationic QAC in neutral conditions.

Structural features of C-PC/QAC-NPs
SEM image revealed that the mean particle size of C-PC/QAC-NPs was about 120nm with spherical morphology (Fig 1).

Drug entrapment efficiency and drug loading efficiency
The drug entrapment efficiency in NPs was measured by spectrophotometer at 650nm and 615nm of C-PC in the supernatant after completely extraction of C-PC from NPs in PBS. Then the total mass of NPs was weighted. Finally, the drug entrapment efficiency and loading efficiency were about 62% and 20%, respectively.

In vitro drug release profile of C-PC/QAC-NPs
The C-PC released from nanoparticles was measured in initial burst in the first 6h and a slow-release pattern as shown in Figure 2. The release rate of C-PC from C-PC/QAC-NPs was 60% in the first 24h cumulatively.
The release pattern could attribute to rapid release of surface adsorbed C-PC and sustaining release of entrapped C-PC from NPs with QAC degradation.

Cytotoxicity studiesg
The cytotoxic effects of different forms and concentrations of C-PC on Hela cells were detected by MTT assay (Figure 3). The results showed that all forms C-PC could inhibit the growth of Hela cells.
Meanwhile, C-PC and C-PC/QAC-NPs induced more toxicity than bare QAC-NPs. The highest concentration (100ug/ml) of C-PC and C-PC/QAC-NPs showed that 40% more toxiticy than bare QAC-NPs on Hela cells.
Thus, it is clearly evident that C-PC/QAC-NPs enhanced the anticancer effect.

Hemolysis assay
It is necessary to make sure the blood compatibility of the drug-NPs, because the drug will finally enter into blood vessels. The hemolysis assay was carried out based on the previous protocol. The optical photographs of blood samples showed clear plasma compared to Triton group (100% hemolysis) with red plasma due to lysed RBCs (Fig 4A). The percentage of hemolysis were less than 5%, the critical safe hemolytic ratio for biomaterials according to ISO/TR 7406 [11]. The results indicated that C-PC/QAC-NPs are hemocompatible.

Discussions
Drug delivery system based on nanoparticle has been become a hot-spot in the field of medicine, which have advantages of high drug-loading efficiency, low adversely effect and sustaining release [12]. Thus, nanoparticles can significantly enhance the drugs to tumor sites and improve their efficacy.
Quaternary ammonium chitosan (QAC), a water soluble chitosan derivative, had an attention in drug delivery application with nontoxic, excellent water solubility and biocompability [13]. MTT assay was adopted to determine the effects of The next step of the study is to link a targeted specific ligands of CD59(CD59sp)on the surface of QAC in order to prepare novel nanoparticals loading C-PC [15].
Making use of the target effect of CD59sp, C-PC is targetly delivered to cancer cells to realize tumor targetting therapy. The study offer a new approach to tumor targeting therapy with wide exploitation and application prospects.

Conclusion
C-phycocyanin loaded quaternary ammonium chitosan were obtained by ionic gelation method. The prepared NPs was about with spherical morphology using SEM.
In vitro drug release showed an initial burst release and sustained release. The C-PC/QAC-NPs showed cytotoxic effects to Hela cells. The C-PC/QAC-NPs were proved to be hemocompatible and safe. In conclusion, QAC increased bioavailability and anticancer property of C-PC. Thus, QAC-NPs were a potential drug delivery system for C-PC into cancer cells.