4.3 Results & Discussions
4.3.1 Preparation, Characterization and Biological Response of RBC- NPs
Figure 4.2 (A, B) Hydrodynamic diameter (A) and zeta potential (B) of the PLGA NPs.
The RBC-NPs were finally prepared by extruding the PLGA NPs along with RBC vesicles through 0.1 µm pore size membrane membrane. In order to optimize the amount of the RBC membrane required to coat PLGA NPs, the RBC-NPs were prepared with different PLGA: membrane protein ratios (1:0.5 to 1:4). Resultant RBC-NPS were subjected to DLS analysis to measure the dH
on Day 0 and 8 in PBS.
Figure 4.3 Hydrodynamic diameters of RBC-NPs prepared with varying PLGA: RBC membrane ratios after synthesis (Day 0) and storage for 8 days.
Results in Figure 4.3 suggested that the PLGA NPs and RBC-NPs prepared at 1:0.5 (PLGA: membrane protein) ratio showed an increase in dH after storage. Nonetheless, RBC-NPs prepared at 1:1 to 1:4 ratio showed greater stability over a period of 8 days, and thus 1:1 ratio was selected for further experiments. Surprisingly, the DLS analyses revealed that the dH of the PLGA NPs was increased from 114 ±10 nm to 157 ±15 nm after RBC membrane coating (Figure 4.4). However, the average size of RBC-NPs was estimated to be 105 ±23 nm from TEM images (Figure 4.5A, B).
Figure 4.4 (A,B) Hydrodynamic diameter (A) and zeta potential (B) of the RBC-NPs.
Additionally, Field-emission scanning electron microscopic (FESEM) images (Figure 4.5D) confirmed the prepared RBC NPs were spherical and possessed almost equal size that was measured in TEM. A possible reason for observation of increased size by DLS measurement, as compared to that of TEM, was due to the hydrodynamic size of the ‘solvated’ NPs being measured.
TEM images of the RBC-NPs, showed a distinct layer on the surface of PLGA NPs confirming the coating with RBC membrane. The surface charge of the NPs plays an important role in their internalization by the cells. In this regard, ζ-potential of RBC-NPs was recorded and shown in Figure 4.4B. The ζ-potential of RBC-NPs was found to be −31 mV, lower than that of bare PLGA NPs (−23 mV, Figure 4.2B), due to successful coating of PLGA NPs with
negatively-charged RBC membrane. The integrity of the proteins in the RBC membrane following their coating onto the PLGA NPs is crucial for the potential interaction of RBC-NPs with the target cells and thus investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Figure 4.5 (A, B) TEM image and Size distribution of the RBC-NPs calculated from TEM images. C: TEM micrograph for PLGA NPs. D: FESEM image of RBC NPs (Size bar: 100 nm).
The image of SDS-PAGE (Figure 4.6) demonstrated that the majority of the proteins from native RBC surface were still present in the RBC-NPs confirming the efficient coating of the PLGA NPs with RBC membrane without loss of the membrane proteins. Taken together, the ability to retain the RBC surface proteins and maintain the size over a period of 8 days confirmed the successful formulation of stable RBC-NPs. Another key point was that the size of the RBC NPs was in accordance with the range required for enhanced permeability and retention (EPR) effect.12
Figure 4.6 SDS-PAGE of the RBC membrane proteins. RBC, isolated RBC membrane and RBC-NPs showed similar protein content confirming the coating of PLGA NPs without loss of the membrane proteins.
Before studying the potential in-vivo application of therapeutic nanoparticles, it is important to check their interaction with the biological system in vitro to ensure safety.13 Therefore, the biocompatibility of the RBC NPs was studied on differentiated macrophage-like THP-1 cells. For this, THP- 1 cells were treated with RBC-NPs and the expression of pro-inflammatory cytokines including IL6, IL8, and IL1β was measured with quantitative realtime PCR. Expression of IL6 stimulates acute phase response and thus it is involved in immune response and inflammation.14 Similarly, production of IL8 attracts the phagocytes to the site of injury or infection, whereas IL1β is also responsible for inflammation.15
Figure 4.7A showed the expression of these cytokines from differentiated macrophage cells after treatment with RBC-NPs and LPS (0.5 µg/mL) as a positive control. From the results, it was observed that the expression of all the cytokines tested was elevated in LPS treatment. However, the expression of
the cytokines was significantly low in RBC NPs treated cells, suggesting the RBC-NPs did not elicit an immune response and thus were biocompatible.
Subsequently, hemocompatibility of the present RBC NPs was tested by incubating them with isolated RBCs. Intact RBCs were isolated and incubated with PBS, RBC NPs and Triton-X (1%) for 3 h at room temperature. From Figure 4.7B, no release of the hemoglobin was observed in negative control (PBS) and RBC NPs samples; whereas significant lysis of the RBCs occurred in the positive control (Triton-X). Collectively, it was concluded that RBC NPs were efficiently stable and did not show any immunogenic or hemolytic response, thus are safe for therapeutic applications.
Figure 4.7 (A) Expression of IL6, IL8, and IL1β in THP-1 cells after RBC-NPs or LPS treatment. (B) % Hemolysis of RBCs in the presence of RBC-NPs and Triton-X. (Data as Mean ±SD, n=3, p<0.0001 denoted as ****).