Development of a Sensitive Assay to Screen Nanoparticles in vitro for Complement Activation

Abstract

Nanomedicines are often recognized by the innate immune system as a threat, leading to unwanted clearance due to complement activation. This adverse reaction not only alters the bioavailability of the therapeutic but can also cause cardiopulmonary complications and death in a portion of the population. There is a need for tools for assessing complement response in the early stage of development of nanomedicines. Currently, quantifying complement-mediated response in vitro is limited due to differences between in vitro and in vivo responses for the same precursors, differences in the complement systems in different species, and lack of highly sensitive tools for quantifying the changes. Hence, we have worked on developing complement assay conditions and sample preparation techniques that can be highly sensitive in assessing the complement-mediated response in vitro mimicking the in vivo activity. We are screening the impact of incubation time, nanoparticle dosage, anticoagulants, and species of the donor in both blood and blood components. We have validated the optimal assay conditions by replicating the impact of zeta potential seen in vivo on complement activation in vitro. As observed in our previous in vivo studies, where nanoparticles with neutral zeta-potential were able to suppress complement response, the change in the complement biomarker was least for the neutral nanoparticles as well through our developed guidelines. These assay conditions provide a vital tool for assessing the safety of intravenously administered nanomedicines.

Graphical Abstract

Introduction

Nanoparticle-based therapeutics, despite their therapeutic promise, can trigger a complement-mediated hypersensitivity reaction that can involve cardiopulmonary problems upon intravenous infusion and be, in some cases, fatal.^1–2 The cardiopulmonary distress ranges from rapid changes in arterial blood gas, vitals and increase blood vessel permeability leading to vasodilation.^3–5 As a result, between 2016 and 2019, only two intravenously delivered nanoparticles have been approved by the FDA and EMA.⁶ This reaction is due, at least in part, to initial intravenous infusion of nanomaterials triggering the complement pathway, a part of the innate immune system.^6–8 The complement system is made up of more than thirty plasma and cell surface proteins.⁹ The complement cascade is activated through three distinct pathways and is responsible for primary surveillance against pathogenic entities.^10–11 The classical pathway relies on complement protein C1 binding to antigen-antibody complex through recognition of the unit C1q, a part of the component C1.¹² Attachment leading to subsequent proteolysis activates the complement proteins C4 and C2 and formation of initial C3 convertase.¹³ The alternative pathway, the second route of activation, occurs due to the spontaneous hydrolysis of C3 further triggered by the presence of foreign surfaces.¹⁴ The third route of activation is through the lectin pathway as plasma protein mannose-binding lectins attach to carbohydrate structures present in the surface of pathogens.¹⁵ Each of the pathways results in the generation of initial C3 convertase that further triggers the pathway into the generation of anaphylatoxins C3a and C5a.¹⁰ The activity of these potent anaphylatoxins range from its pro-inflammatory functions in degranulation of mast cells and production of histamines,^{12, 16} vasodilation, increased permeability of blood vessels¹⁷ as well as its regenerative roles in tissue restoration,¹⁸ and neuronal development.^19–20 The complement system is the first-in-line defense of the immune system against pathogens and active at all times controlled by several complement regulators.^{11, 21} However, if the activation is not inhibited efficiently, it leads to prolonged tissue damage and glomerular disease along with the inflammatory responses mentioned above.¹⁰ As the complement system is directed towards attacking any pathogenic or foreign substance, nanomedicines are reported to elicit complement-mediated initial infusion reaction as well.

When nanoparticles are administered intravenously, they are engulfed with complement and serum proteins. The complement protein C3b either binds directly to nanoparticle surfaces or attach to the serum proteins adsorbed on the surface of the particles, with the attachment being reversible and dynamic.^22–23 C3b deposition on nanoparticles along with factor Bb and Properdin in presence of Magnesium ion will lead to complement activation and further amplification of C3b.^22–23 This infusion-reaction driven complement-mediated response,^{5, 24} is of concern for nanoparticle systems broadly including liposomal nanoformulation, Doxil,^25–26 inorganic nanoparticles such as iron oxide and metallic nanoparticles used as contrast agents for imaging,^27–28 organic nanoparticles such as poly(lactic-co-glycolic acid)-b-poly(ethylene glycol) and polystyrene^{24, 29–31} based nanoparticles. It has been challenging to characterize the complement early in development, as different species show different levels of sensitivity, and the response generated in vivo in large animals is difficult to resolve in vitro.^{4, 32}

The three most common methods for measuring complement activation are indirect measurement through hemolysis assay,^33–35 quantifying the proteins that attach to the nanoparticle surface,^{23, 26–27, 36–37} and through enzyme immunoassays.^{4, 28} These methods use nanoparticle blood interaction to generate complement proteins, and then the proteins that attach directly or indirectly on the surface of nanoparticles is quantified.^{23, 27–28} Hemolysis assays such as CH50 assays and AH50 assays are used to indirectly measure the extent of activation by measuring the amount of serum that causes lysis of 50% of sheep erythrocytes due to exposure of serum to complement activator.³⁴ However, the method can include spontaneous hemolysis and variable affinity of the to-be-lysed erythrocytes.³³ Thus methods like hemolysis assays are often semiquantitative.³⁸ Some of the recent complement studies involve incubating blood matrices with nanoparticles and quantifying the attached complement proteins through SDS-PAGE and western blots.^{23, 27, 36} Moreover, the protein corona is dynamic due to reversible attachment of proteins, and this can lead to underestimating the overall complement protein levels, mainly quantifying the complement proteins part of the hard corona.³⁹

Enzyme-linked immunoassays (ELISAs) for detecting complement have been available for more than two decades, and the assays are more sensitive than CH50 assays,⁴⁰ but ELISAs are only available for limited species. Moreover, the enzyme immunoassays do not consider interference in signal outputs due to the presence of nanoparticles in the blood, plasma, or serum. Nanoparticles, because of their physiological properties and morphology, may lead to adsorption of assay reagents or biomolecules generating outputs with disturbance.⁴¹ Nonetheless, the sensitivity of the enzyme immunoassays makes these extremely attractive by offering the possibility of developing in vitro assays that replicate the in vivo response allowing efficient screening of molecular features of nanomaterials and their impact on complement activation. We hypothesized that by optimizing the particle dosage, species of blood matrix, and anticoagulants,⁴² we would be able to develop a set of conditions in vitro that correlated with our previous in vivo findings and would allow efficient screening of nanomaterials. To test this hypothesis, we investigated the impact of blood matrix conditions, time, and dilution factors to replicate the sensitive response seen in vivo in an in vitro assay.

Experimental Section

Materials

Assay conditions were developed using heparinized human whole blood, plasma, and complement protected human serum, and heparinized porcine whole blood and plasma obtained from Innovative research Inc (Novi, MI). Porcine citrated plasma and human citrated plasma were also obtained from Innovative research Inc (Novi, MI). Zymosan was obtained from Sigma Aldrich. Dulbecco’s phosphate-buffered saline (without phosphate and magnesium) was obtained from Fisher Scientific. C5a human ELISA duo kit (DY2037) was obtained from R&D systems (Minneapolis, MN). Human Complement C3 ELISA Kit (ab108823) and Guinea Pig Complement C3 ELISA Kit (ab157705) were obtained from Abcam plc (Cambridge, MA).

Nanoparticles prepared for validating the assay involved l-lactide from Polysciences Inc (Warrington, PA), heterobifunctional poly(ethylene glycol) with 5000 Da molecular weight from Laysan Biosciences (Arab, AL), and d-lactide from Purac Biomaterials (Corbion, Amsterdam, Netherlands). All solvents used were ACS grade and obtained from Fisher Scientific.

Methods

Developing complement assay conditions

Plasma and serum were stored at −80°C until used for the experiments and were thawed by placing them in a 37°C water bath. Whole blood samples were stored at 4°C until used. As a positive control, zymosan, a known complement activator,⁴³ is used. To 500ul aliquots of blood matrices, 100ul of zymosan suspended in Dulbecco’s PBS (without calcium and magnesium) (PBS) was added and pipetted gently for mixing. As a negative control, PBS without zymosan was used.

Initially, the role of anticoagulant in blood collection was assessed. The response generated for heparinized and citrated human blood plasma was determined by incubating the samples with zymosan at 1mg/ml dosages for 30 minutes. Next, the differences in complement response in porcine and human blood samples were also investigated for choosing the appropriate species for mimicking the reaction in vitro. The optimum incubation times were then determined by incubating the samples with the nanoparticle suspension at 37°C for 30, 45, and 60 minutes, respectively. The response in single donor vs. pooled blood samples was also compared for human serum and heparinized human plasma and whole blood. After incubation, the samples were centrifuged at 4000g for 5 minutes. The serum or plasma was aliquoted in clean tubes and stored on ice until the assay was carried out. Dilutions of the supernatant serum or plasma were prepared, and the assays were carried out following the protocol for the ELISA assay (C5a ELISA assay duo kit, R&D Systems). For immunoassay quantifying biomarker C5a, the optical density for the samples and standards were measured at a wavelength of 450 nm using SpectraMax M2 Microplate Reader (Molecular Devices LLC) with background correction done using reading obtained at 540 nm. Human C3 ELISA kit and guinea pig C3 ELISA kit from Abcam plc. were used for quantifying the biomarker C3.

The fold change was measured by comparing the level of C5a observed in the sample incubated with PBS and the sample with zymosan.

$$\text{Fold change}=\frac{\text{Biomarker in sample containing zymosan}-\text{Biomarker in sample containing PBS}}{\text{Biomarker in sample containing PBS}}$$

Validating assay’s sensitivity to mimic in vivo response in vitro

The developed complement assay was used to determine whether the complement response generated due to differences in surface charge of nanoparticles could be detected precisely. The method of preparing the nanoparticles are summarized in the supplementary data. The nanoparticles fabricated were thoroughly characterized through dynamic light scattering for determining the size and zeta potential using Malvern Zetasizer Nano ZS ((Malvern Panalytical, Malvern, UK).

The ELISA conditions used were identical, as mentioned above. Heparinized human whole blood was incubated with the nanoparticles of various zeta-potential at a dosage of 0.25mg/ml. As a positive control, zymosan, a known complement activator,⁴³ is used at the same dosage. Aliquoted whole blood in amounts of 500ul was incubated with 100ul of nanoparticles suspended in Dulbecco’s PBS (without calcium and magnesium). The samples were incubated at 37°C for 45 minutes and then centrifuged at 4000g for 5 minutes to separate the nanoparticles. The separated plasma was aliquoted in clean tubes and stored on ice until the assay was carried out. The fold-change in each of the samples is quantified comparing the quantity of C5a in whole blood incubated with PBS only to the amount of C5a in samples with nanoparticles or zymosan.

$$\text{Fold change}=\frac{\text{C}5\text{a in sample containing zymosan}/\text{nanoparticles}-\text{C}5\text{a in sample containing PBS}}{\text{C}5\text{a in sample containing PBS}}$$

Nanoparticles used for validating the assays

Poly(lactic-co-glycolic acid)-b-poly(l-lysine)-b-poly(ethylene glycol) (PLGA-PLL-PEG) nanoparticles were obtained from the previous study on the response of the nanoparticles in vivo in a porcine trauma model.³¹ Three formulations retrieved from the previous study were used in this study, where two of the formulations had peptide cRGD conjugated to the particles. The particles were stored at −20°C and characterized through DLS, ensuring the particle size did not change significantly over the storage time. The zeta-potential was also recorded for these PLGA-PLL-PEG nanoparticles. All protocols involved with the previous study on porcine liver injury model³¹ were in accordance with animal protocols reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at Case Western Reserve University. The protocols were developed by Gurney et al⁴⁴ and modified in conjunction with the Trauma Research Laboratory at Massachusetts General Hospital.

Poly(lactic acid)-b-poly(ethylene glycol) (PLA-PEG) nanoparticles with varied zeta-potential were prepared and characterized through DLS. The methods for synthesis, fabrication, and characterization of the nanoparticles are included in supplementary data.

Statistical Analysis

One-way ANOVA was used to determine the statistical significance of the difference of C5a in samples containing nanoparticles and zymosan, compared to samples with PBS. As post-analysis, Dunnett’s multiple comparison test was used for comparing to the control group PBS, and Tukey analysis was used for comparing the means for determining which groups displayed statistically significant differences. For donors or blood samples comparing two groups, a t-test was used to determine whether the means were significantly different.

Results

Comparing complement response observed in citrated and heparinized human blood plasma

Two commonly used anticoagulants for collecting blood samples were compared to investigate their impact on complement activation. We analyzed the complement protein C3 which is cleaved when complement activation happens via any of the pathways leading to a short term decrease in C3.^{10–11, 21} Zymosan, a known complement activator,⁴³ was used to generate the response. Heparinized blood matrices preserved C3 more than the citrated matrices (supplementary figure 1). This makes heparin the more attractive choice as an anticoagulant because it retains the greater complement response. This result is not entirely surprising. The complex correlation between the coagulation cascade and complement pathways is a significant factor in generating the response as coagulation components thrombin and factor Xa can directly activate components of the complement cascade.⁴⁵ The choice of anticoagulant is essential, to ensure that complement cascade is not activated or blocked as an aftermath. Of the commonly used anticoagulants, citrate prevents coagulation by sequestering Ca²⁺, while heparin acts by enhancing antithrombin activity.⁴⁵ EDTA, another commonly used anticoagulant, acts by chelating Ca²⁺ and Mg²⁺ entirely, and as a result blocks the complement cascade as well, as the metal ions are essential components for the activation.⁴⁵ Heparin, as an anticoagulant at lower dosages, has been found not concomitantly to impact the complement cascade.⁴⁶ As a result, the rest of the experiments involved utilizing heparinized blood plasma and whole blood for porcine as well as human donors.

Impact of incubation time in generating complement response in human blood matrices

We investigated the ideal incubation time for generating a complement response. The biomarker quantified was the complement protein C5a. In heparinized plasma, we incubated zymosan at concentrations ranging from 1mg/ml to 5mg/ml. The incubation time for generating the response is dependent on several factors. A computational model addressing time-dependent depletion of C5 and generation of C5a-desArg exhibit an upward trajectory up to 25 minutes, with the rate of change gradually decreasing.⁵² The zymosan suspended in phosphate-buffered saline was added to the plasma and incubated for 30, 45, and 60 minutes respectively at 37°C in a rotating shaker. Incubation for 45 minutes showed a concentration dependent response to zymosan (Figure 1). in Hence, for further experiments, the incubation time of 45 minutes was chosen for quantifying the change with the greatest sensitivity.

Figure 1:

Validation of incubation time at 45 minutes. Statistical analysis using one-way ANOVA shows the fold-change at 5mg/ml dosage for 45 minutes incubation is significantly different from the fold-change for the same time-point at 1mg/ml dosage.

Differences in complement response generated in human blood, plasma, and serum

We subsequently investigated the role of different blood components, i.e., blood plasma, serum, and whole blood obtained from human donors in generating the complement response. The groups include single donor heparinized blood plasma, pooled heparinized blood plasma, single donor serum, and pooled complement-protected serum obtained from human donors. The groups were incubated for 45 minutes at 37°C in a rotating shaker. Incubating with zymosan at dosages of 0.25mg/ml leads to an increase in C5a as expected due to complement activation in all the groups. The fold-change was calculated relative to the amount of C5a detected in groups incubated with phosphate-buffered saline (Fig 2). While the fold-change in single donor heparinized plasma was 20-fold, the change in pooled heparinized plasma was 5-fold (Fig 2 B and C). The fold-change in the single donor serum was 12-fold, while pooled complement protected serum had a 14-fold increase (Fig 2 D and E). The anaphylatoxin C5a is generated as the complement cascade advances. The baseline upon incubation with PBS is different for single donor and pooled plasma samples. This difference is attributed to the difference in complement levels among different donors. On the other hand, in serum, as the coagulation cascade is no longer intact, complement protein levels are high due to initial activation followed by the coagulation cascade, and the level change is faster compared to plasma.⁵³

Figure 2.

Comparing complement response among single donor and pooled plasma and serum. A. Incubation of blood plasma and serum with zymosan at a dosage of 0.25mg/ml and quantifying complement protein C5a. The C5a levels in the samples are quantified through enzyme immunoassay, and the fold change is determined with respect to the amount of C5a determined in the serum or plasma incubated with PBS. B. The change in complement protein C5a in single donor heparinized plasma resulted in a fold change of 20±4 fold. C. The change in complement protein C5a in single donor serum resulted in 12±2 fold. D. The complement protein C5a changed by 5±0.4 fold in pooled heparinized plasma. E. The complement protein C5a changed by 14±1 fold in pooled serum. Using t-test, the means observed for PBS and zymosan were compared for each sample and the means were significantly different.

Therefore, from the responses observed from the fold changes for C5a, we can compare the complement response in different matrices. As complement and coagulation cascades often have cross-interaction^{50, 54–55} among themselves, we see a consistent response for fold change in C5a in the pooled and single-donor serum, compared to plasma. Hence, in comparison to plasma, the serum is more suitable for generating the complement response in vitro.

The in vitro response in whole blood was investigated to further assess the suitable form of blood product for simulating in vivo complement response in vitro. We studied the impact of the concentration of zymosan by quantifying changes due to zymosan at a concentration as high as 1mg/ml, as well as a lower concentration of 0.25mg/ml (Fig 3). The whole blood was incubated with zymosan suspended in phosphate-buffered saline at the mentioned dosages for 45 minutes at 37°C while shaking. The complement protein C5a was quantified in the samples. At a concentration was lowered to 0.25mg/ml, the fold-changes observed were 14-fold, 31-fold, and 20-fold in samples collected from three different donors. (Fig 3 B, C, and D). While we see a high fold change for C5a for 1mg/ml zymosan, even for the same dosage of 0.25mg/ml, whole heparinized blood showed higher complement response compared to both plasma and serum. Based on the response and higher sensitivity, whole heparinized blood is the most suitable matrix, followed by single-donor and pooled serum for these assays.

Figure 3.

Complement response in heparinized whole blood. A. Schematic of process. B. Fold change and complement level in donor 3. C. Fold change and complement level in donor 4. D. Fold change and complement level in donor 5. The changes due to a dosage of 0.25 mg/ml where 14±6 folds, 31±1.7 and 20±1.9, respectively. Using t-test, the means observed for PBS and zymosan were compared for each donor and the means were significantly different.

We sought to develop a sensitive assay that would be able to simulate the complement response observed in vivo using blood products in vitro. On comparing whole blood, plasma, and serum, the response generated in whole blood is found to be more sensitive with higher fold changes observed on incubation with Zymosan. Moreover, based on the fold-changes observed, the dosage of 0.25 mg/ml of Zymosan resulted in generating a sensitive response, highlighting the differences in complement response from donor to donor.

Validating impact of zeta-potential of nanoparticles observed in vivo through developed in vitro assay

Based on our observations, we chose the incubation time of 45 minutes and dosage of 0.25mg/ml of zymosan or nanoparticles to investigate further the impact of the nanomaterials in generating a complement response. The impact of dosage of nanoparticles on the complement response are discussed in the supplementary information (Figure S5). The nanoparticles were incubated at 37°C with whole heparinized blood for the incubation time in a rotating shaker (Fig 4). The samples were then prepared following the assay protocol for quantifying the amount of complement protein C5a after incubation with the nanoparticles, positive control zymosan, and negative control phosphate-buffered saline at the dosage of 0.25mg/ml.

Figure 4.

Method for generating complement response in vitro. Heparinized whole blood is incubated with nanoparticles suspended in PBS at dosages of 0.25mg/ml for 45 minutes at 37℃ in a rotating shaker. The samples are centrifuged to separate the nanoparticles and the supernatant serum is used to quantify the complement activation biomarker C5a through immunoassay.

The objective of our work was to develop an in vitro assay that can mimic the complement response observed in vivo. As a first step, we investigated whether the developed assay can capture the response observed in vivo for poly(lactic-co-glycolic acid)-b-poly(l-lysine)-b-poly(ethylene glycol) (PLGA-PLL-PEG) nanoparticles in a porcine trauma model. We have previously seen that PLGA-PLL-PEG-based hemostatic nanoparticles generate complement response upon infusion in porcine trauma models as well as upon naïve administration.³¹ When the nanoparticles were infused intravenously, the nanoparticles lead to exsanguination in minutes.³¹ While slow infusions can help overcome such hypersensitivity reactions,⁵⁶ even lower dosages lead to complement activation.³¹ Moreover, the hemostatic nanoparticles augment hemostasis by binding with GPIIb/IIIa integrin of activated platelets and subsequently increase survival, and the formulation needs to be administered in bolus to reach targeted efficacy.^57–58 As highly negative⁵⁹ or highly positive surfaces charge of nanoparticles³⁰ can lead to complement mediated hypersensitivity reactions, the role of tuning zeta-potential was investigated. The study showed that nanoparticles with zeta-potential between −3.0 mV and 3.0 mV were able to prevent the hypersensitivity reactions at lower dosages (below 3.3 mg/kg).³¹ We tracked the impact of three such PLGA-PLL-PEG nanoparticles on cardiopulmonary vitals and the rate of blood loss upon infusion in porcine animals. The formulations include the PLGA-PLL-PEG-1 without any peptide motif conjugated to it, and two other formulations PLGA-PLL-PEG-CRGD-1 and −2 with same properties but fabricated in different batches. The fabricated nanoparticles were in the neutral range but administering at higher dosages (3.3 and 6.6 mg/kg) had led to complement activation in vivo. The formulations PLGA-PLL-PEG-cRGD-1 and PLGA-PLL-PEG-1 lead to exsanguination at 10- and 5-minutes post-infusion showing moderate signs of cardiopulmonary distress, were the nanoparticles were infused at t=5 minutes (i.e. 5 minutes post-injury). (Fig 5 A–H). From figure 5, mild cardiopulmonary distress is visible from the change in heart rate compared to baseline measurements at t=0 minutes as well as abrupt changes in end-tidal CO₂, indicative of respiratory distress and shock.⁶⁰ While the formulation PLGA-PLL-PEG-cRGD-2 did not lead to immediate exsanguination, moderate respiratory as well as cardiac distress was visible post-infusion. In all three infusions, the rate of blood loss increased at 5–10 minutes, i.e., after infusion of nanoparticles, indicating vasodilation and increased permeability leading to subsequent blood loss. When the complement proteins were quantified in the blood samples from the porcine animals receiving the PLGA-PLL-PEG nanoparticles through infusion, there were no significant differences in complement protein C3 and C3a.³¹ However, there was an increase in the C5a level.³¹ Once complement pathway is activated, the complement protein C3 upon complement activation would deplete, leading to the eventual generation of anaphylatoxins C3a and C5a, where the anaphylatoxins are responsible for cardiopulmonary distress, vasodilation, increased permeability of blood vessels.^{16, 20} Thus, from the in vivo response due to the infusion, keeping in mind the complexity of trauma upregulating complement⁶¹ as well, we see moderate complement response from the neutral nanoparticles at high dosages.³¹ This upregulation is mainly from the increased blood loss after infusion, possibly due to anaphylatoxins like C5a acting as vasodilators. Most importantly, the lack of complement detection at the molecular level, even in case of moderate hypersensitivity response observed in vivo, highlights the need to develop robust assays for quantifying complement response with high sensitivity.

Figure 5.

Change in cardiopulmonary vitals upon infusion of PLGA-PLL-PEG nanoparticles in swine. Infusion of the nanoformulation PLGA-PLL-PEG-cRGD-1 resulted in increased rate of blood loss and fluctuations in the vitals with exsanguination at 10 minutes post-infusion. A. Change in heart rate, B. Change in end-tidal CO₂, C. Change in blood pressure, D. Change in rate of blood loss due to PLGA-PLL-PEG-cRGD-1 infusion. Infusion of the nanoformulation PLGA-PLL-PEG 1 increased rate of blood loss and led to exsanguination at 5 minutes post infusion. E. Change in heart rate, F. Change in end-tidal CO₂, G. Change in blood pressure, H. Change in rate of blood loss due to PLGA-PLL-PEG-1 infusion; Infusion of the nanoformulation PLGA-PLL-PEG-cRGD-2 increased rate of blood loss slightly without causing exsanguination, however, fluctuations were exhibited in the vitals. I. Change in heart rate, J. Change in end-tidal CO₂, K. Change in blood pressure, L. Change in rate of blood loss due to PLGA-PLL-PEG-cRGD-2 infusion. These data are from a previous study looking at the impact of modulating zeta potential on the complement response to hemostatic nanoparticles in a porcine model of trauma.³¹

As first step of validating our assay, we determined how the complement protein C5a changes upon incubation with the PLGA-PLL-PEG nanoparticles. The nanoformulations mentioned above resulted in 3.3±0.2 folds change for PLGA-PLL-PEG-cRGD-1, 3.4±0.4 folds for PLGA-PLL-PEG-1, and 4.4±0.5 folds for PLGA-PLL-PEG-cRGD-2 (Fig 6). The differences in the fold changes were statistically significant compared to the response seen in sample incubated with the negative control, phosphate-buffered saline (PBS). While an increase in complement protein itself does not always lead to a hypersensitivity reaction, a two-four folds increase in terminal complement proteins is observed in the blood samples for cases showing clinical symptoms of complement-mediated hypersensitivity reaction for PEGylated liposomal doxorubicin.^{5, 26} Thus, the developed assay can interpret the molecular response in vitro upon incubation with nanoparticles that had shown moderate complement activation in vivo based on the observed fold change in complement protein C5a upon incubation of the PLGA-PLL-PEG nanoparticles with whole heparinized human blood.

Figure 6.

Fold change in complement protein C5a due to incubation of whole blood with PLGA-PLL-PEG nanoparticles. One-way ANOVA followed by Tukey analysis shows that while the means of fold changes are significantly different for the nanoformulations compared to Zymosan, the difference is not statistically significant for the changes within the three groups PLGA-PLL-PEG-cRGD-1, PLGA-PLL-PEG-1 and the PLGA-PLL-PEG-cRGD-2.

To further validate the responsive assay conditions, we chose to study the difference in complement response in whole blood due to nanoparticles having different zeta potential, as zeta potential, a surface property related to the surface charge of nanoparticles is known to impact complement activation.^{31, 59} We designed nanoparticles with a range of zeta potentials from highly negative to neutral (Fig 7 A and B). The hydrodynamic diameter of the nanoparticles, Z-average, and the zeta potential was determined using dynamic light scattering (Fig 7 C and D). Details of the methods for synthesis, fabrication, and characterization of the nanoparticles are included in supplementary data and supplementary figures 3 and 4. The polymers poly(l-lactic acid)-b-poly(ethylene glycol) with terminal amino and carboxyl groups were used to fabricate the nanoparticles. Nanoparticles with zeta-potential ranging from −1.8 mV to −23.5 mV were prepared (Fig 7 D). The nanoparticles have a poly(lactic acid) core, with polyethylene glycol corona on the surface. Adjusting the ratios of the amino and carboxyl groups on the surface helps in controlling the zeta-potential of the nanoparticles.

Figure 7.

Characterization of nanoparticles with varied zeta-potential. A. Nanoparticles with poly(lactic acid) core and poly(ethylene glycol) corona. B. Varying amino and carboxyl groups to vary zeta-potential. C. Hydrodynamic diameter of nanoparticles. D. Zeta-potential of nanoparticles. 3400PEG indicates PEG with a molecular weight of 3400 Da wile 5000PEG indicates a molecular weight of 5000 Da for the PEG units, respectively.

To validate the assay using the fabricated nanoparticles with a range of zeta-potential, the fold-changes in the incubated samples were determined compared to samples incubated with phosphate-buffered saline. For highly negative zeta-potential, −23.5mV and −15.6 mV, the fold-change was highest, 1.9-fold. As the zeta potential moved towards the neutral range, i.e., between −3.0 mV and 3.0 mV, the fold-change in C5a also decreased. The observations agree with the reduced complement activation due to the neutral zeta-potential of nanoparticles seen in vivo. The fold change is lowest for the neutral nanoparticles at −1.8 mV showing a fold change of −0.02-fold (Fig 8.). There is no statistically significant difference between the C5a levels for PBS and neutral nanoparticles at −1.8 mV. The exchange of carboxyl groups on the exposed nanoparticle surface with amine groups while fabricating the neutral nanoparticles leads to an increase in zeta-potential on the surface resulting in neutral zeta-potential. The lower change in complement protein C5a in whole heparinized blood incubated with these neutral nanoparticles signifies that the complement cascade is not highly activated suggesting that the complement cascade is not treating the incubated nanoparticles as pathogens and trying to clear it out of the system immediately. Whereas the upregulated C5a levels based on the fold change observed for the negative nanoparticles at −23.5 mV and −15.6 mV indicate that the complement system is treating the nanoparticles as a threat and working on the removal of the nanoparticles. Initially, C3b and iC3b binds to the foreign surface, preparing the foreign agent for subsequent clearance,^{23, 62–63} while liberating potent anaphylatoxins and fragments that work in formation of the eventual terminal membrane attack complex. As previously mentioned, two to four-fold change in complement protein is indicative of probable signs of complement-mediated hypersensitivity reaction and related clinical symptoms in vivo.^{5, 26} Surface charge is a parameter relevant for complement activation,^64–66 and through controlling zeta-potential a closely related parameter to surface charge, the complement response can be controlled.³¹ The designed assay conditions were able to validate the complement response in vitro from the perspective of neutral zeta-potential of nanoparticles and its role in evading complement response. While in vivo studies often show only clinical signs and symptoms of hypersensitivity reactions as manifestations of complement activation with the absence of a robust molecular response for the complement proteins itself, we developed an assay that can generate the complement-mediated response in vitro mimicking the response associated with changing zeta-potential of nanoparticles.

Figure 8.

Impact of zeta potential of nanoparticles on complement response. Neutral nanoparticles lead to decreased complement activation in vitro leading to lower fold change in complement protein C5a. However, aggregation of particles with a low zeta potential led to an increase in complement protein C5a.

Besides validating the impact of zeta-potential, we quantified the changes in C5a due to aggregation. Aggregated nanoparticles with a zeta-potential of −4mV resulted in fold change of C5a (2.3±0.05) equivalent to that observed in more negative unaggregated nanoparticles (2.3±0.05 fold) (Fig 8 and 9). Increasing zeta potential towards neutral range can reduce the change in C5a; however, the aggregated nanoparticles close to neutral range lead to a higher change in C5a compared to unaggregated nanoparticles of similar zeta-potential of −5.7 mV (Fig 8) and C5a comparable to that of the −15.6 mV unaggregated nanoparticles (Figure 9) suggesting the significance of size and aggregation in generating the hypersensitivity reaction as well. Size and curvature of nanoparticles are as a result essential to control complement alongside zeta-potential, as these parameters dictate how the convertase molecules would interact with the surface.^67–68 Aggregation of the nanoparticles can lead to higher deposition of C3b, a complement protein essential in forming initial convertase in the complement cascade, as seen for polypropylene sulfide nanoparticles of different size.⁶⁹ Thus, using the assay conditions, we were able to generate the complement response dependency on surface charge as well as size and morphology of nanoparticles in vitro.

Figure 9:

Impact of aggregation of particles. The complement activation as marked by C5a is increased with aggregated particles at levels similar to unaggregated particles with a high zeta potential relative to the whole blood with PBS control.

Discussion

Because of the lack of sensitive assays to quantify complement-mediated response in vitro, we worked on developing assays that would be able to detect complement changes in vitro with precision and sensitivity, as observed in vivo. To determine the optimum assay conditions, we narrowed down the factors that play a critical role in generating the complement response. We chose heparinized blood and blood matrices as citrate and EDTA act as chelators for Ca2+ and Mg2+ inhibiting the activation of the complement pathway.^{45, 70} The extent of complement activation is species-dependent, hence while developing assays to quantify changes in complement protein level, a significant challenge is selecting the right species that is hypersensitive. For a liposomal formulation that is known to cause complement activation at a dosage ranging from 0.01–0.3mg/kg in porcine animals, a dosage of 5–25mg/kg is required to get a similar response in rodents and a dose of 0.05–0.1mg/kg in canines. Whereas for reactive human species, which accounts for around 7% of human population, the required dose is comparable to that observed in porcine animals, i.e., 0.01–0.2mg/kg.⁴ Moreover, the blood matrix selected while designing assays, i.e. whole blood vs. plasma vs. serum, is also crucial, due to interplay between complement and coagulation cascades and its impact on the complement protein levels as well as anticoagulant used during a blood draw as that can affect the complement pathway as well.^{45, 70} Complement activation is a dynamic process as the reaction can start in seconds, and hence while designing assays, the role of incubation time plays a critical role as well. Therefore, while developing assays to measure complement activation accurately, factors that play a crucial role include the donor of species, form of blood sample (i.e., plasma, serum, and whole blood), the anticoagulant for collecting blood, the dosage of the nanoparticle, and incubation time for generating the response in vitro.

As a control, we have used zymosan for tuning the complement assays. Zymosan can activate the complement system through the alternative pathway as the insoluble yeast cell wall component is able to deplete C3 in presence of Mg²⁺ and properdin.⁷¹ The surface of zymosan provides immune adherence scope for the complement fragment C3b and enhances phagocytosis.⁷² Complement activation due to biomaterials is dependent on surface coating densities and moieties that impacts covalent binding with the complement protein C3b^{23, 73}, and binding of C3b with the plasma proteins IgG and human serum albumen^74–75 that immediately engulf the nanoparticles in the blood stream.²³ As our nanoparticles are PEGylated, and based on correlation between PEGylation density and complement activation pathways⁷³, we suspect complement activation through the alternative pathway to be the significant pathway of activation as the nanosurfaces provide scope of C3b and iC3b binding and subsequent activation.²³

The complement system in pigs and humans are different. Pulmonary intravascular macrophages (PIM), key components of the mononuclear phagocyte system, are responsible for non-IgE mediated hypersensitivity reactions in pigs.⁷⁶ However, the PIMs are morphologically similar to Kupffer cells⁷⁷ in the human liver that are responsible for generating multiple complement receptors and clearance of nanoparticles.⁷⁸ The key difference between the two is how the response is distinct in different species. In comparison to Kupffer cells, PIMs release significant quantities of vasoconstrictors and complement mediators that are responsible for respiratory distress and inflammation.⁷⁶ As a result, porcine in vivo models are highly sensitive. However, as only a subset of human population shows the sensitivity, this naturally raises questions on the relevance of the porcine model. But one must also look at the purpose behind using the porcine in vivo model as a guiding tool. The goal of the porcine model is to determine whether a nanomaterial leads to unwanted hypersensitivity reaction at a subtherapeutic dosage.⁷⁹ Thus, as a tool for confirming the safety of a nanoformulation in hypersensitive patients, the porcine in vivo model is essential.

However, it is also essential to note that complement factors such as anaphylatoxin receptors can bind with the anaphylatoxins as well.⁸⁰ Time and dosage both are critical in this respect, especially for highly sensitive species such as the pigs. In our assessments using porcine plasma, we see a decrease in complement protein C5a with increasing dosage of complement activator zymosan with increasing incubation time. This could be explained due to the porcine complement system being more robust and generating a response in a short period, especially for a component like C5a that has lower half-life in vivo.³⁸ That is, whether the split products in porcine blood samples have a higher propensity in interacting with inhibitors compared to the split products in human blood samples need to be further evaluated. Currently, the in vitro assessment of complement biomarkers by immunoassays is highly limited in porcine blood and its matrices. Most often, in vitro assessments involve using western blots,⁸¹ and very few immunoassays have been documented in the literature for use in vitro. While the response can be tracked through cardiopulmonary vitals observed in vivo in porcine matrices^{5, 82}, generating the response in vitro needs to be worked on as a critical step before moving to in vivo studies.

While several immunoassays have been developed over the years for quantifying complement, none of them considers interference due to nanoparticles in the blood, plasma, or serum. Nanoparticles itself may lead to adsorption of assay reagents or biomolecules and interfere with the assay outputs.⁴¹ Thus, the samples need to be extensively washed and appropriately groomed to avoid interference in the outputs for the actual complement level detections. The biomarkers quantified are the molecules C3 and the fragment C5a generated due to the depletion of C3. On the event of complement activation, once initial C3-convertase is formed, the convertase catalyzes the breakdown of C3 into the smaller fragment C3a and larger fragment C3b.^{10–11, 21} The pathway proceeds leading to the generation of the anaphylatoxins and eventual terminal complex that works on clearing the threat out of the system.^{10–11, 21} As there is a lack commercially available enzyme immunoassay for different species, the C3 ELISA kit for guinea pig was used to track changes in complement protein in vitro in the porcine plasma. This kit was previously used in quantifying changes in vivo in the porcine trauma model from the blood samples drawn.³¹ C5a, one of the anaphylatoxins generated, is a potent inflammatory molecule that targets immune and non-immune cell receptors and leads to vasodilation, increased cell permeability, and contraction of smooth muscles.¹⁰ While there are immunoassays for quantifying another potent biomarker C3a, the ELISA assay required subsequent acid-base neutralization for removal of whole proteins from plasma before quantifying C3a des Arg (Complement C3a des Arg Human ELISA Kit, ab133037) which could lead to degradation of nanoparticles within the samples while processing. Hence, using the biomarker C5a for detection served as a simplified tool for quantifying complement response in vitro.

We determined that heparinized human whole blood has higher sensitivity for generating complement response in vitro. Compared to plasma, serum shows higher sensitivity as well. This is due to the absence of cells leading to biological turnover, and as a result, the complement pathway is not suppressed in the absence of the coagulation cascade.^{38, 83–84} The differences in fold changes in whole blood due to incubation with zymosan at 0.25 mg/ml can be explained by the difference in responsiveness among individuals, as blood samples are collected from different donors. However, in all cases, there is significant complement activation. For complement split products, patients who show signs of hypersensitivity reaction show at least a two-four fold^{5, 26} increase compared to baseline for PEGylated liposomal doxorubicin. All the whole blood donors showed a more than ten-fold increase in C5a in vitro. Hence, the incubation time of 45 minutes for whole heparinized blood at dosages of 0.25mg/ml for complement activator, i.e., either zymosan or nanoparticles were chosen as an optimum scenario for simulating the in vivo complement response.

As steps for validation, we generated the response observed in vivo in porcine animals for PLGA-PLL-PEG nanoparticles through the in vitro assay using human blood. While porcine animals are more susceptible to complement activation compared to humans,^{4, 85} the pathway of activation, and subsequent response is analogous. The manifestations of complement-mediated hypersensitivity reactions, especially the changes in biomarkers, impacts on cardiopulmonary vitals, along with the time-course of the activation are similar in both humans and porcine animals, with reaction frequency being the point of difference between the two species.⁸⁵ A thorough characterization of the porcine C7 complement protein, one of the complement protein that contributes in the formation of the terminal membrane attack complex in the activated complement pathways, shows that the protein exhibits comparable structural and functional characteristics with the human complement protein C7⁸⁶ and same complement inhibitors can inhibit the catalytic activity of the C5 convertase in human and porcine models in vitro and in vivo.⁸⁷ The proteins share epitopes across species with cross-reactivity to same detection antibodies,⁸⁸ but the response mostly occurs at different rates, requiring optimization. Moreover, detection antibodies are species-specific and that acts as a limiting factor especially for the commercially available ELISA assays, as such assays could essentially help in safety assessments of the nanomedicines before moving to large animal studies.

While the hypersensitivity response generated due to the PLGA-PLL-PEG nanoparticles is robustly visible in vivo, in vitro assay did not show strong complement activation previously. Intravenous administration of highly positive and negative poly(lactic-co-glycolic acid)-b-poly(l-lysine)-b-poly(ethylene glycol) nanoparticles can lead to complement-mediated hypersensitivity reactions, especially in large animals.³¹ For the same nanoparticles, controlling zeta-potential leads to control over the complement response at a specific range of dosages.³¹ Most importantly, tracking the complement proteins in vivo did not exhibit any significant change, but the rapid change in blood gas and vitals highlight the hypersensitivity response due to infusion. Through the developed assay, the complement protein C5a change was found to be more than three-fold for all the formulations. As we have mentioned in the previous section as well, patients with visible hypersensitivity reaction symptoms exhibit at least a two-four fold^{5, 26} increase in the complement biomarker.

Zeta-potential is a parameter related to surface charge and relevant for complement activation.^64–66 The fact that nanomaterials can elicit such innate immune response is not surprising because of the sheer surface area exposed to complement proteins. A 400-nm particle can provide a surface area of 5×10⁵ nm². Further introducing functional groups,^{69, 89} and charged moeities³⁰ helps in controlling zeta potential on the surface and subsequent interaction with complement convertase molecules as well. Thus, tuning the surface properties can have an impact on complement activation and the infusion reaction. Once we mimicked the in vivo porcine complement response using whole human blood in vitro, we applied our developed assay conditions to simulate the change in complement response due to the changing zeta potential of nanoparticles observed in vivo.^31,64 Nanoparticles were generated with different zeta potential with the polymer poly(lactic acid)-b-poly(ethylene glycol). The zeta-potential observed for the nanoparticles is generated due to the ratio of surface moieties present. We quantified the anaphylatoxin C5a in the blood samples post-incubation and found that the response was least for neutral nanoparticles with zeta-potential of −1.8 mV. The observations confirm what we had seen while investigating the role of zeta-potential in complement response in vivo in porcine animals.³¹ Moreover, the designed assay conditions were able to simulate how aggregation and nanoparticle morphology⁶⁹ also dictates interaction with the convertase molecules on the exposed foreign surface. Using the optimum assay conditions determined, we designed an in vitro screening tool that can generate reactogenicity observed in vivo validated through simulation of complement activation due to zeta-potential and size of nanoparticles.

Conclusion

Highly sensitive immunoassays for quantifying complement response are essential in designing safer nanomedicines. Being able to screen nanomaterials before moving into large animal models, or, potentially, being able to replace large animal models with effective in vitro assays has the potential to greatly increase the number of nanomaterials that can be translated to the clinic. We have developed an assay with high sensitivity for evaluating complement response in vitro. This is supported by our observations on simulating in vivo complement response observed in our previous work³¹ in vitro as well as further assessment of the impact of zeta-potential on complement-mediated hypersensitivity reactions. We believe this optimized assay provides a new method to effectively screen nanomaterials for complement activation so that we can more effectively design nanomaterials that have high safety profiles and the potential to transform medicine.