Effect of Surface Modification and Macrophage Phenotype on Particle

Article pubs.acs.org/Biomac

Effect of Surface Modification and Macrophage Phenotype on Particle Internalization Daniel Wang,† Ngoc Phan,‡ Christopher Isely,‡ Lucas Bruene,‡ and Kaitlin M. Bratlie*,†,‡,§ †

Department of Materials Science and Engineering and ‡Department of Chemical and Biological Engineering, Iowa State University, Ames, Iowa 50011, United States § Ames National Laboratory, Ames, Iowa 50011, United States S Supporting Information *

ABSTRACT: Material properties play a key role in the cellular internalization of polymeric particles. In the present study, we have investigated the effects of material characteristics such as water contact angle, zeta potential, melting temperature, and alternative activation of complement on particle internalization for pro-inflammatory, pro-angiogenic, and naiv̈ e macrophages by using biopolymers (∼600 nm), functionalized with 13 different molecules. Understanding how material parameters influence particle internalization for different macrophage phenotypes is important for targeted delivery to specific cell populations. Here, we demonstrate that material parameters affect the alternative pathway of complement activation as well as particle internalization for different macrophage phenotypes. Here, we show that the quantitative structure− activity relationship method (QSAR) previously used to predict physiochemical properties of materials can be applied to targeting different macrophage phenotypes. These findings demonstrated that targeted drug delivery to macrophages could be achieved by exploiting material parameters.

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INTRODUCTION

material properties and the biological system is crucial to improve targeted drug delivery. Rational design of polymers for drug delivery or tissue engineering remains a challenge that many researchers are attempting to overcome.15,16 One of the ways to improve rational design would be through mathematical models generated by QSAR as has been recently applied to biomaterials.17−19 This method allows for modeling linear relationships between alternative activation of complement with multiple surface modifier parameters, thus, allowing for an understanding as to which material properties influence the measured value. As a temperature-responsive “smart” polymer, poly(Nisopropylacrylamide) (pNIPAm) has been extensively studied for applications in drug delivery due to its biocompatibility and its phase transition at 32−34 °C that results in a volume decrease, allowing for ease of drug loading.20,21 Here, we synthesized p(N-isopropylacrylamide-co-acrylic acid) (p(NIPAm-co-AAc)) particles and modified them with 13 different functional groups to investigate the effects of chemical ̈ moieties on particle internalization in M1, M2, and naive macrophages. We use LPS and IL-4 to induce M1 and M2 macrophages, respectively. Correlations between particle internalization and material properties were examined.

Macrophages are the main scavengers responsible for the clearance of particulate debris, fluid, and foreign substances in the immune system.1,2 They exist on a continuum of phenotypes,3 which arise from the different microenvironment cues present in the host. In response to signals derived from microbes, solute or activated lymphocytes, macrophages undergo polarization or reprogramming,4 which leads to the expression of various cell-surface receptors for cellular uptake, cytokine and chemokine secretion profiles, and so on. For simplicity, we classify macrophages into two polarization states in our work: the inflammatory or classical activated M1 macrophages and anti-inflammatory or alternatively activated M2 macrophages. Tumor-associated macrophages are thought to be M2 cells, and present an attractive target for drug delivery in which alternatively activated macrophages could be reconditioned to destroy malignant cells.5,6 In principle, both material properties and the biological system are significant to regulating particle internalization. Material properties include both physical properties, such as particle size, shape, and deformability, and chemical properties, which depend on functional group.7−11 Previous studies have shown that the geometry of the particles can have strong effects on phagocytosis.7 Modeling studies on particle internalization have also demonstrated the effects of the cell cytoskeleton and ligand density on phagocytosis.12−14 However, in these studies, a library of functional groups for understanding the effects on particle internalization was not evaluated. The impact of macrophage phenotypes on particle internalization was also not explored. Improved understanding of the relationship between © XXXX American Chemical Society

Received: August 4, 2014 Revised: September 29, 2014

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dx.doi.org/10.1021/bm5011382 | Biomacromolecules XXXX, XXX, XXX−XXX

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2.4. Zeta Potential. Milli-Q water was neutralized to pH 7 with HCl or NaOH to ensure that the ions in water would not interfere with the zeta potential of particles. A low salt concentration was confirmed by measuring conductivities 0.5 is good and Q2Y > 0.9 is excellent.26 Kubinyi has stated that R2 ≥ 0.81 for in vitro data should be used to state that the data is good.27 Additionally, the difference between R2Y and Q2Y should not be larger than 0.2−0.326,28 for a good fit. Unsurprisingly, increasing the hydrophobicity of the materials synthesized here caused complement to hydrolyze (R = −0.767). This is in agreement with previous reports in which protein adsorption reaches a maximum at a WCA of ∼50° 29 and complement is activated by hydrophobic materials.30 The observation that increasing the number of

fluorescenceLPS = 2.11 × 103 + 2.98 × 101N + 7.21 × 101NC + 4.44 × 101NH + 1.30 × 102NCH2 − 6.60Ndc + 5.40 × 101WCA E

(3a)


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Figure 5. continued

F


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̈ macrophages. (A) The fluorescence level of FITC labeled particles internalized by Figure 5. Particle internalization by LPS or IL-4 activated or naive ̈ macrophages, respectively. Data represents the mean value of three replicates for each sample ± standard deviation. LPS or IL4 activated, or naive ̈ macrophages. Scale bar: 50 μm; * indicates p < Representative images for each particle internalized by (B) LPS or (C) IL-4 activated, or (D) naive 0.05 compared to p(NIPAm-co-AAc) for that particular macrophage activation.

(R = 0.692), which likely contribute to the hydrophobicity/ hydrophilicity of the particle. Ndc, a term that includes structural parameters or atomic correction terms, has a negative impact on LPS stimulated macrophages (R = −0.286) and a ̈ cells (R = 0.246). The IL-4 positive impact on the naive activated macrophages also have a strong positive correlation with the number of sp2 carbon atoms (R = 0.931), which is likely tied to the complement activation since this trend was observed for complement activation in Figure 6. Complement activation was not included in the QSAR analysis of particle internalization for the three macrophage treatments since the goal was to develop a model for predicting internalization using material properties. The correlations were R = −0.345, 0.716, ̈ macrophages. The and −0.320 for the LPS, IL-4, and naive negative correlation between % lysis for the LPS activated and naiv̈ e macrophages is expected since complement forms opsonins when cleaved.36,37 The positive correlation between % lysis by complement for IL-4 actiaved macrophages indicates that internalization is increased when complement is not activated through the alternative pathway. It has been found that IL-10 protects cells from complement-mediated lysis,38 which is up-regulated for IL-4 activated macrophages.6 There are also correlations with the number of nitrogen atoms (R = 0.800) and Ngroup (R = 0.600) for IL-4 stimulated macrophages. Ngroup accounts for polar functional groups. This finding likely results from the increased surface charge on the particle surface, which is known to influence phagocytosis.39−42 In a similar vein, more negatively charged particles also resulted ̈ macrophages in a higher likelihood of internalization by naive (R = 0.251). This is not surprising since surface chemistry is known to influence opsonization, which ultimately activates the macrophage response.43 Furthermore, surface charge has a significant impact on particle internalization. There was also a slight dependence on H-bond acceptors (R = 0.309). A negative impact was observed from the melting temper̈ macrophages only (R = ature of the particles for the naive −0.501), meaning that softer particles were more readily internalized. This is inline with previous findings that material rigidity plays a role in internalization by macrophages.44

Figure 6. Plot of predicted % lysis of red blood cells from alternative activation of complement vs experimentally determined (observed) values. Error bars represent the standard deviations of the observed and predicted values. The results for the statistics of the model were the following: Q2Y = 0.829 and R2Y = 0.871.

fluorescenceIL4 = 4.35 × 103 + 3.05 × 102Nsp2 + 5.13 × 102NN + 1.37 × 102NC − 1.88 × 102NCH2 − 2.92 × 102NO + 4.66 × 101Ngroup

(3b)

fluorescencenaive = 4.37 × 103 + 1.31 × 102HA − 1.21 × 102NO − 6.76 × 101Tm + 5.86 × 101ζ + 4.89Ndc + 2.72 × 102WCA

(3c)

In building eq 3, the correlations between the material descriptors and the measured value were used to evaluate the importance of the descriptors and the measured internalization. Interestingly, different material descriptors impacted particle ̈ internalization dissimilarly for the three activations. Both naive and LPS activated macrophages exhibited a slight positive correlation with WCA (R = 0.481 and 0.533, respectively). LPS-activated macrophages were very positively correlated with the number of hydrogen atoms (R = 0.727), the number of CH2 groups (R = 0.739), and the total number of carbon atoms G


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̈ macrophage internalparticles can positively affect the naive ization process. The generalities uncovered in this work pave the way for rational design for drug delivery to distinct macrophage phenotypes.

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ASSOCIATED CONTENT

* Supporting Information S

NMR spectra and loading efficiency of the nanoparticle. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: 515-294-7304. Fax: 515-294-5444. E-mail: kbratlie@ iastate.edu. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Science Foundation under Grant No. CBET 1227867 and the Roy J. Carver Charitable Trust Grant No. 13-4265. The authors also acknowledge support from NSF ARI-R2 (CMMI-0963224) for funding the renovation of the research laboratories used for these studies.

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REFERENCES

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Figure 7. Plot of predicted fluorescence vs experimentally determined (observed) values for particle internalization. Error bars represent the standard deviations of the observed and predicted values. (A) LPS treated cells. The results for the statistics of the model were the following: Q2Y = 0.794 and R2Y = 0.853. (B) IL-4 treated cells. The results for the statistics of the model were the following: Q2Y = 0.847 ̈ cells. The results for the statistics of the and R2Y = 0.924. (C) Naive model were the following: Q2Y = 0.813 and R2Y = 0.939.

In total, there are several parameters that are linked to specific macrophage phenotypes, implying that targeted delivery of macrophages can be achieved through exploiting material parameters. Specifically these parameters are melting ̈ macrophages; increased sp2 carbon atoms temperature for naive for IL-4 activated macrophages; and number of hydrogen atoms, number of carbon atoms, and number of CH2 groups for LPS activated macrophages.

5. CONCLUSIONS The material characteristics, particle internalization, and biochemical inhibitory data reveals interesting findings. First, the alternative complement pathway can be affected by hydrophobicity, the number of CH2 groups, and the number of sp2 carbon atoms. Second, distinct material parameters influence particle uptake for different macrophage phenotypes. Particle internalization of LPS activated macrophages can be engineered by changing the surface charge, the number of hydrogen atoms, and the number of 2° carbon atoms. Decreased complement activation and increased sp2 carbon atoms positively impacts cellular uptake for IL-4 activated macrophages. Decreasing the melting temperature of polymeric H


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Effect of Surface Modification and Macrophage Phenotype on Particle

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