Influence of PEG chain on the complement activation suppression and longevity in vivo prolongation of the PCL biomedical nanoparticles
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- Shan, X., Yuan, Y., Liu, C. et al. Biomed Microdevices (2009) 11: 1187. doi:10.1007/s10544-009-9336-2
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The process of opsonization is the major biological barrier to the injectable polymeric nanoparticles (NPs). Complement protein is one kind of opsonins and it can be activated potentially by the negative charged particles. The fragment C3b generated by complement activation could subsequently induce the opsonization on the NPs surface. The aim of our work was to examine the relationship between the hydrophilic poly(ethylene glycol) (PEG) chain on the surface of NPs and particles longevity in vivo from the biological point of view such as complement activation (C3 cleavage) as well as uptake by macrophages. The studies showed that the introduction of PEG chains led to slightly smaller NPs with lower polydispersities than those prepared from naked poly(ε-caprolactone) (PCL) and enhanced the ζ potential of NPs from −27.17 mV to −6.046 mV. It was also found that PEG hydrophilic chain could decrease the C3 cleavage and remarkably suppress opsonization and phagocytosis subsequently. In biodistribution investigations in vivo, as a control, PCL NPs were present in MPS tissues in the first 5 min followed by metabolism elimination rapidly, whereas the PEGylated NPs had more particles blood retention in vivo after injection. In fact, in present work, it has been convinced that these results in vivo could be predicted by the in vitro fluorescent phagocytosis model and the extent of complement activation in advance.
KeywordsPEGylation NPsProlong longevity in vivoBiodistributionComplement activation
Blood substitutes based on hemoglobin created for potential use in human have attracted comprehensive attention due to the limited supply of fresh blood (Teramura et al. 2003; Lou Carmichael 2001; Chang 1999; Vadapalli et al. 2002; Sou et al. 2007). However, with regard to intravenous biomaterials device including blood substitutes, the reticuloendothelial system (RES) is the most determinative biological barrier to predominantly limit the injected polymeric NPs efficacy due to its highly efficiency in eliminating the circulating particles from blood stream as foreign objects (Chambers 2004) following opsonization of plasma protein on particles surface. Considerable studies have reported that, for biological applications involving intravenous administration of particles, size distribution and surface characteristic are the most important parameters influencing the ultimate fate of the particles in body. (Yague et al. 2008) In detail, freely floating particles greater than 200 nm can be physically trapped by the fenestrations in the spleen (Yague et al. 2008; Storm et al. 1995; Moghimi and Davis 1994; Klibanov et al. 1991), while the sub−70 nm-sized NPs favor to accumulate in liver. In short, the promising longevity NPs in blood should be in 70–200 nm size range.
Meanwhile, previous researches have revealed that the NPs with highly hydrophobic surface (Lee et al. 2007; Carstensen et al. 1992) or positive charge (Dong and Feng 2004) are also very affinity to the opsonic proteins. Several methods of camouflaging or masking NPs (NPs) have been employed for particles surface modification through physical or chemical attachment in terms of neutralize the NPs surface charge (Yague et al. 2008) or provide a repulsive steric barrier (Voss et al. 2007) to prevent the flocculation of particles and decrease opsonization by blood components (Ogawara et al. 2001; Moghimi et al. 2001). Poly(ethylene glycol) (PEG) has uncharged hydrophilic characteristic, and high steric stabilization with non-toxic resulting in dramatically changing the charge of biomaterials, which thus has been a major strategy to decrease the non-specific interactions of complexes with serum components, and thereby increase blood circulation time. (Sato et al. 2007; Torchilin 2007)
In our previous study (Zhao et al. 2007), PEG chains were introduced into hydrophobic poly(ε-caprolactone) (PCL) NPs to improve the hydrophilicity of NPs and prolong the longevity of polymeric NPs in blood stream. Results showed that, after intravenous (i.v.) administration, the longevities of PCL-PEG NPs in blood circulation were prolonged approximately 7.2-fold as long as that of the naked PCL NPs.
In that study, however, we only found this excited phenomenon without any mechanism analysis: “Why PEGylation could prolong the NPs longevity in blood stream?” After intravenous administration, the charged NPs will interact with blood components and generate the complement activation in serum. Therefore, it is important to investigate the PEGylation NPs in terms of triggering the complement activation and what is the superior function of PEG hydrophilic chain on prolonging the biomedical device longevity in vivo. Furthermore, the direct association between the hydrophilic PEG chains at the surface of NPs and the biological properties (such as complement system and the phagocytosis behavior) has not yet been investigated.
In present work, we compared the physicochemical characteristics (such as the size distribution and the ζ potential) of the PCL NPs and PCL-PEG NPs. Secondly, the amount of C3 activated by NPs was quantitatively determined to assess the contribution of the complement system to NPs accumulation into organs and blood circulation in vivo and in vitro. Subsequently, the in vitro macrophage uptake, in vivo biodistribution following i.v. administration of NPs labeled by fluorescence 6-coumarin were analyzed. To mimic the phagocytosis in vivo, the primary culture of mouse peritoneal macrophages (MPM), a classical phagocytic cell line model (X.Q.Shan et al. 2009), was selected to carry out the in vitro macrophage uptake experiment. Meanwhile, the biodistribution of these two types of labeled NPs were evaluated by calculating the NPs accumulation in major organs (heart, lung, liver, spleen, and kidneys) in order to trace the site of particles in vivo at different time-points and verify the prediction of phagocytosis in vitro.
Lyophilized Bovine hemoglobin (Hb) was purchased from YuanJu Biotechnology Company (Shanghai, China). The NPs were prepared with PCL (MW45k) and PCL-PEG (MW42k, MW PEG:MW PCL = 1:6), which were obtained from Chengdu Institute of Organic Chemistry, Chinese Academy of Science (Chengdu, China). The particles were labeled by Coumarin-6(Sigma,St. Louis,,MO, USA). The stabilizer poly (vinyl alcohol) (PVA1750), the emulsifiers Span80 and the solvent ( methylene chloride, acetic ether and acetone) were all of analytical grade obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). ICR mice (25 ± 1 g in weight) were obtained from Shanghai Animal Center, Chinese Academy of Science (Shanghai, China).
2.2.1 Preparation of Hb-loaded PCL and PCL-PEG NPs
Hb-loaded PCL and PCL-PEG nanopartcles were both formed by double emulsion (W/O/W) and the solvent diffusion/evaporation technique as previously described (Zhao et al. 2007). In brief, 10 mg polymer, 6-coumarin (10 μg) and 0.15 g Span80 were dissolved in mixture of methylene chloride, acetone and acetic ether (5 ml) as organic phase. Then an aqueous solution of hemoglobin (0.15 g/ml, 0.5 ml) was emulsified in the organic phase using a probe sonicator (JYD-900, ZhiXin Instrument Co., Ltd., Shanghai) (50 W for 15 s) to form a primary oil-in-water emulsion. This initial emulsion was further mixed in the stabilizer of PVA-containing aqueous solution to make a w/o/w double emulsion with a high-pressure homogenizer. After that, the double emulsion was poured into 110 ml water solution and the system was stirred for the removal of the solvent under atmospheric pressure at room temperature.
2.2.2 Physicochemical characterization of the particles
Approximately 100 mg NPs were re-dispersed in 10 ml PBS (pH7.4) for several minutes using an ultrasonic bath. The size distributions and zeta potentials of the NPs suspensions were determined at 25℃ by Dynamic Light Scattering (DLS) using Zetasizer Nano ZS (Malvern Instruments Ltd., UK).
2.2.3 Evaluation of the complement activation C3
Clinically, the cleavage extent of complement component C3 was a measure of the extent of complement activation (Kainthan et al. 2006). Complement activation evaluations were carried out as follows: 1 ml of fresh healthy human serum was mixed thoroughly with 0.1 ml of NPs suspension which was containing 15 mg of lyophilized hemoglobin NPs. Equal volumes of pure serum and PBS solution were incubated at 37℃ for 30 min, respectively. The samples C3 concentration were determined by a Behring Nephelometer 100 analyzer and calculated using a standard curve.
2.2.4 NPs in vitro uptake by mononuclear phagocytes
Phagocytic uptake of NPs was determined by using murine peritoneal macrophage cell harvested from ICR mice (weight in 25 ± 1 g). The 1 ml Mercaptoacetate was injected into the abdominal cavity of mice. 30 min later, the mice were sacrificed and the peritoneal cells were collected by flushing the peritoneal cavity with 10 ml of 0.9% NaCl. Then, the MPM were isolated by centrifuged at 1000 rpm for 5 min and re-suspended in complete culture medium. After transferred to 24-well plate (1 ml for well), the cells were incubated at 37°C in atmosphere containing 5% CO2 for 24 h. The purification of macrophages by washing with culture medium to remove non-adherent cells; adherent cells were further incubated in culture medium.
At the end of this incubation time, 100 μL of coumarin-6 labelled NPs isotonic Na chloride suspension (150 g/l) were incubated with the MPM at 37°C for period of 30 min. The phagocytized particles were observed using a fluorescence microscope (Nikon TE2000, Nikon Instruments, Melville, NY).
2.2.5 Biodistribution study of PCL and PCL-PEG NPs
The tissues distribution of PCL and PCL-PEG NPs labeled with coumarin-6 following intravenous administration to ICR mice (25 ± 1 g) was determined as we previously in detail (Zhao et al. 2007). Briefly, the mice were injected in tail vein with 10 ml/kg (body weight of mice) of NPs suspension 150 mg/ml. At 5 min, 30 min, 2 h, 6 h, 24 h and 48 h after injection, plasma samples were collected from fossa orbitalis. Subsequently, the mice were sacrificed; the organ samples, consisting heart, liver, lung, spleen and kidneys were removed, quickly rinsed and weighted. The biodistribution was assayed for coumarin-6 extracted by acetonitrile by the spectrofluorimeter at λex485nm and λem538nm to estimate the amount of the NPs in each organ. For calculation, standard curves of NPs in every organ were prepared by addition of coumarin-6-labeled NPs suspension to each organ at different concentration gradient following the same treatment steps.
3.1 Characterization of NPs
The effects of PEGylation on NPs physicochemistry characteristics
Size distribution (nm)
ζ potential (mV)
183.4 ± 10.7
0.188 ± 0.029
−27.17 ± 2.92
159.6 ± 15.8
0.159 ± 0.020
−6.046 ± 3.76
But the surface electrical potential of the NPs varied with the PEGylation pronouncedly (Table 1). NPs obtained were both negatively charged, and the ζ potential enhanced from −27.17 mV to −6.046 mV with the PEG-modification, indicating that the PEG chains shielding around the surface of NPs. (Mosqueira et al. 2001b)
3.2 Evaluation of the PEG effect on the complement activation C3
Complement can be non-specifically activated by biomaterials resulting in local and system effect such as platelet aggregation and inflammatory reactions (Cenni et al. 2008). Usually, the foreign surface activates the complement through the classical pathway and the alternative pathway, both of which leading to activation of C3 and then initiating the opsonization. And it was mentioned that in certain circumstance, the PEG can trigger the complement activation (Hamad et al. 2008). Therefore, in our study, the effect of NPs prepared by the two types of polymer on the complement were evaluated through investigated the concentration of C3.
3.3 Evaluation of the PEG effect on the phagocytic uptake of NPs in vitro
3.4 Evaluation of the PEG effect on the NPs biodistribution in vivo
PCL-PEG NPs showed initial higher blood circulation levels than PCL NPs did. An obvious retardation in clearance from the blood could be observed during the initial 2 h after intravenous administration. Therefore, 48 h after injection, a larger amount of fluorescent labeled PCL-PEG NPs were still found in the blood stream, and its half-life time (160 min) was 7.2-fold as long as that of PCL NPs. (Reviewed in Ref.19)
Additionally, the majority of the PCL NPs accumulate in liver at 24 h after intravenously injection with 57.58% of the injected dose. In contrast, 10.50% of the PCL-PEG NPs initial dose was recovered from the liver; and the concentrations in else organs investigated seem low. In detail, for the injected PCL-PEG NPs, the percentage of the total dose, accumulated in the heart (1.65%), lung (1.22%), and kidneys (5.61%) was a little higher than for the PCL NPs (0.35%, 0.63% and 4.27%, respectively).
In this work, the physicochemical properties, the complement activation, phagocytic behavior and the biodistribution properties of PCL-PEG NPs were investigated. Conventional PCL NPs were also included in this study for comparison reasons. The PCL-PEG NPs had ζ-potential values relatively close to neutral owing to the presence of PEG with electrical neutrality on their surface which covers the surface charges, whereas the PCL NPs had a highly negative ζ-potential. Moreover, the size of PCL-PEG NPs was smaller than that of the PCL NPs. This may be considered to be an indirect evidence of the micellar-like structure of the PCL-PEG NPs prepared in this study (Win and Feng 2005).
It was well known that the intravenous NPs were quickly removed from blood circulation within several minutes. This problem was the real challenge faced by investigators who focused on the intravenous drug delivery system. To address this problem, many studies have highlighted that the hydrophilic chains on the NPs surface formulated a hydrated layers to decrease hydrophobic interactions between the protein and the surface (Vonarbourg et al. 2006) which resulting in prolonging the circulation time in blood. Okuda et al (Okuda et al. 2006) had reported that PEGylated amino acid dendrimers substantially enhanced the particles blood retention in vivo. Li et al (Li et al. 2001) described that the PEGylation could extend half-life of BSA loaded in PLGA NPs from 13.6 min to 4.5 h. In our previous study, it has been also found that the half-life time of PCL-PEG NPs was prolonged approximately 7.2 times longer than the naked PCL NPs. (Reviewed in Ref.19)
However, why PEGylation could prolong the NPs longevity in blood stream? Actually, after injected in blood stream, these foreign products would contact with blood cells and the endothelium immediately, and then may induce an array of biological responses. These responses had some direct or indirect relevant with phagocytosis. Accordingly, in this paper, we will discuss and answer this question from this biological point of view.
Following injection into the bloodstream, the long-circulating intravenous biomaterials NPs, especially the blood substitutes, would be removed rapidly resulting from the phagocytosis. The phagocytosis is an irreversible process with mainly three steps: ①recognized by opsonins such as the complement protein, immunoglobulins G and M, fibronectin and apolipoproteins, or by specific or non-specific receptors present at the surface of the macrophage plasma membrane (Vonarbourg et al. 2006); ②then adsorbed by these opsonins; ③finally, eliminated by the mononuclear phagocyte system (MPS). That means if we could prevent the complement protein from activation, the phagocytosis may be inhibited subsequently.
From the immunological point of view, C3, as a major complement protein, plays a significant role in complement system. After opsonization, cleavage of C3 by C3 convertase would be activated into a large fragment of C3b and a small fragment of C3a. C3b provides specific recognition by type CR1 receptor on macrophages (Mosqueira et al. 2001b), thus the evidence of C3 cleavage was obtained by quantified the C3 concentration, which was used as a way to characterize the avoidance of complement activation by NPs. Some studies found that the negative charge on the particles surface may be a potential activator of human complement system. (Moghimi and Hunter 2001) Therefore, the PEG chains on the NPs surface neutralized the zeta potential, as shown in Table 1, resulting in suppressing complement activation. On the other hand, it could be conformed that the brush-like PEG chains may sterically prevent the deposition of C3b onto the surface of NPs (Gbadamosi et al. 2002) and reduce interaction with phagocytic cells membrane (Mosqueira et al. 2001b). Our results, demonstrated in Fig. 1, showed that the PCL NPs were prone to cause complement activation. On the contrary, compared with naked PCL NPs, the PEG copolymers had a lower degree in C3 cleavage than the negative control PBS did.
In short, biological studies in present work showed the significant PEG effect on complement consumption of PCL-PEG NPs, in comparison with pure PCL NPs. Therefore, it could be hypothesized that the PEGylation NPs could avoid uptake by mononuclear phagocyte more effectively. Fortunately, it was convinced by our study on NPs uptake by MPS in vitro. As shown in Fig. 2, significant decreases of the NPs with emitting much weaker fluorescent light by phagocyte were observed with the absence of PEG. Fig. 3 shows that compared to the PLA NPs, the internalization in organs of PEGylated NPs decreased significantly in vivo.
The in vivo biodistribution data accompanied with the evidence of uptake by macrophages showed substantial difference in phagocytosis uptake between PCL and PCL-PEG NPs. During the formation of PCL-PEG NPs, the hydrophobic chain of PCL can incorporate with each other, while the hydrophilic chain of PEG chain, remaining on the surface, protrude to the outer phase and form a conformational hydrophilic “cloud-layer” over the NPs that protect the particles from each other and various plasma proteins. As a result, the PEGylation NPs could avoid uptake by MPS.
Indeed, the most marked inhibition of phagocytosis was owing to the hydrophilicity and steric effect of PEG chains, which ensure the particles a longer half-life time in blood stream.(Reviewed in Ref.19)
The cardiovascular NPs longevity in blood stream could be affected by many aspects of their physicochemistry characteristics. Therefore, it is necessary to consider the basic properties such as size distribution and ζ potential and biological properties in order to have a clear picture of the way of NPs do to the half-life time. The available physicochemistry results suggest that PEG chains binding to hydrophobic PCL backbone could remarkable neutralized the surface charge of naked PCL NPs.
The biologic studies showed that the sterically PEG hydrophilic chain could protect the NPs from activating the complement system, which resulting in surface opsonization by the fragment C3b generated by complement activation.
Although the description of the adsorption protein is unknown, these observations imply that, under the protection of PEG chains to NPs, quiescent macrophages are not sufficiently activated yet by some blood component adsorb on NPs surface. It could be predicted by an in vitro phagocytosis model. Finally, the in vivo biodistributions of these two types of NPs, respectively, are investigated to verify this prediction and support the conclusion that the PEGylation indeed avoid uptake by MPS tissues in order to prolong the NPs circulation time in blood stream.
The authors acknowledge the financial support from the National High Technology Research and Development Program of China (863 program) (No. 2004AA-302050) and from Shanghai Nanotechnology Special Foundation (No. 0452 nm022).