Homotypic targeting of immunomodulatory nanoparticles for enhanced peripheral and central immunity

Abstract Objectives Synthetic oligodeoxynucleotides (ODNs) that contain unmethylated cytosine–phosphate–guanine (CpG) motifs serve as immune adjuvants in disease treatment. However, the poor cell permeability and safety concerns limit their medical applications, and biocompatible strategies for efficient delivery of functional CpG ODNs are highly desirable. Materials and Methods Self‐assembled, cell membrane‐coated CpG nanoparticles (NP) are prepared, and their physicochemical properties are characterized. The uncoated and membrane‐coated CpG NP are compared for their biocompatibility, cellular uptake kinetics, endocytic pathways, subcellular localization, and immunostimulatory activities in macrophages and microglia. Results Macrophage‐ or microglia‐derived cell membrane camouflaging alters the endocytic pathways of CpG NP, promotes their targeted delivery to the cells with homologous membrane, ensures their endosomal localization, and enhances their immunomodulatory effects. Conclusions We design a type of biomimetic NP consisting of self‐assembled CpG NP core and cell membrane shell, and demonstrate its advantages in the modulation of peripheral and central immune cells. Our study provides a new strategy for the application of CpG ODNs.


| INTRODUC TI ON
Unmethylated cytosine-phosphate-guanine (CpG) dinucleotide motifs belong to a classic type of pathogen-associated molecular patterns (PAMPs) and exist extensively in the genomic DNA of bacteria and viruses. [1][2][3][4] The recognition of exogenous CpG motifs is mediated by the Toll-like receptor 9 (TLR9) of host cells. 3,5,6 As a class of host innate immune response, subsequent TLR9 activation primes a series of signal transduction, at least in part via the nuclear factor κ-light-chain enhancer of activated B cells (NF-κB) pathway and the mitogen-activated protein kinase (MAPK) pathway, inducing the expression of proinflammatory cytokines such as tumor necrosis factorα (TNFα) and interleukin-6 (IL-6). [7][8][9] Synthetic oligodeoxynucleotides (ODNs) comprised CpG motifs are found to stimulate similar immunomodulatory responses and display a great potential in the therapy of infection, cancer, or allergy. 5,10,11 However, free CpG ODNs are susceptible to nuclease cleavage and have poor cell permeability due to their anionic and hydrophilic properties. [12][13][14][15][16] Although phosphorothioate modification of the backbone can confer the resistance to nuclease degradation, it concurrently increases the cytotoxicity and thereby limits the clinical utilization of CpG ODNs. [17][18][19][20] Therefore, it is highly appealing to develop efficient and safe delivering strategies for CpG ODNs, improving their bioavailability and biocompatibility.
Nanoparticles (NP) have been widely explored as carriers of small molecule chemicals and biomolecules. [21][22][23][24][25][26] Recent studies have demonstrated their superior advantages in the delivery of functional nucleic acids including short interfering RNA (siRNA), viral RNA mimics, and CpG ODNs. As an example, a tumor microenvironment-responsive nanobooster encapsulates a hybrid RNA encoding both siRNA and viral RNA signature for orchestrated activation of innate immunity and potent induction of long-term T cell memory, eliciting multilayer antitumor activities. 27 Many types of nanodevices have been designed for the loading of CpG ODNs.
Among them, Gold NP represent one kind of convenient option as they can form stable interaction with CpG ODNs containing either thiolated terminal or polyadenine sequence. 19,28 Besides, CpG ODNs can be easily tethered to DNA nanostructures via complementary base pairing. 29 Despite such progress, nanocarriers that aim for more efficient packaging and precise delivery of CpG ODNs are still in great demand.
Although nanocarriers are often modified with targeting molecules as a guidance to specific organs or tissues, the formation of protein corona on the surface of NP can compromise the delivery efficiency. [30][31][32][33] Recently, camouflage with natural cell membrane has become an advanced strategy for surface functionalization of NP. [34][35][36][37][38][39] Cell membrane-coated NP possess many unique properties of the source cells, especially the capacity of homotypic targeting that allows for active accumulation of NP in their desired destination. In addition, surface coating with cell membranes can greatly enhance the biocompatibility and circulation lifetime of NP and promote their cellular uptake. [40][41][42] Herein, we utilize one-step self-assembly of CpG NP, which are further camouflaged with the cell membrane of macrophages or microglia, for targeted immunomodulation in peripheral or central immune system ( Figure 1A). Compared to uncoated CpG NP, the Raw264.7 macrophage cell membrane-coated NP (CpG NP@ M R ) and the BV-2 microglial cell membrane-coated NP (CpG NP@ M B ) display significantly increased levels of cellular internalization.
In addition, the cell membrane coating switches the endocytosis of CpG NP from a clathrin-dependent pathway to caveolae-and macropinocytosis-dependent pathways. Nevertheless, the majority of cell membrane-coated CpG NP are delivered to the endolysosomal compartments, allowing for TLR9 recognition and subsequent signaling activation. Accordingly, both CpG NP@M R and CpG NP@ M B stimulate higher expression levels of proinflammatory cytokines (TNFα and IL-6) than uncoated CpG NP in macrophages and microglia, respectively ( Figure 1B).

| Preparation and characterization of CpG NP
CpG ODNs and PAH were utilized to assemble CpG NP via crosslinking with genipin. In brief, 2-mg CpG ODNs and 9-mg genipin were dissolved in 2.8-ml Milli-Q water and stirred for 24 h. The mixture was diluted to 14 ml, slowly dripped into 10-ml PAH solution (Mw = 15 kDa, 0.3 mg/ml), and further stirred for 24 h. The assembled CpG NP were purified by centrifugation and washing with Milli-Q water. Agarose gel electrophoresis was utilized to confirm the formation and stability of CpG NP. The hydrodynamic diameter and zeta potential analysis were carried out on a ZEN3690 Zetasizer (Malvern).

| Cellular internalization of CpG NP and CpG NP@M
To study the cellular uptake of CpG NP and CpG NP@M,

| Cytokine assays
Raw264.7 or BV-2 cells were dispersed at a density of 0.5 × 10 5 cells per well in 24-well plates and treated with isolated cell membranederived vesicles, CpG ODNs, CpG NP, or CpG NP@M (CpG ODNs: 2 µg/ml). After incubation for 48 h, the supernatants were collected and centrifuged at 13,523 g for 10 min. The purified supernatants were used for ELISA assays following the protocols provided by the manufacturers.

| Statistical analysis
The results were presented as mean ± standard deviation (s.d.) or standard error of the mean (s.e.m.) from three or more independent samples. The Student's t-test was used to determine the statistical significance of the differences between two groups.

| Preparation and characterization of cell membrane-coated CpG NP
CpG ODNs and PAH were utilized to fabricate the self-assembled CpG NP via crosslinking with genipin. The stability of self-assembled CpG NP was tested in a gel retardation electrophoresis. Free CpG ODNs migrated in the agarose gel in a time-dependent manner, whereas the CpG NP were retained in the well without the detection of dissembled CpG ODNs ( Figure S1). The hydrodynamic diameter of CpG NP was ~140 nm, with a positive surface charge (33.9 ± 4.7 mV).

| Cellular uptake efficiency
Coating with cell membrane can confer NP with homotypic affinity that is mediated by adhesive interactions between specific cell surface proteins, enhancing the binding of camouflaged NP to the same type of cells. 43,44 We thus speculated that the coating of macrophage membrane would promote the cellular uptake of CpG NP by

| Endocytic pathways
The different internalization kinetics of CpG FAM NP and CpG FAM NP@ M R triggered our interest to explore whether the membrane coating and Figure S5B). These results suggested that the camouflage with cell membrane shifted the endocytosis of CpG NP from a clathrinmediated pathway to caveolae-and micropinocytosis-mediated pathways.

| Intracellular trafficking
The TLR9 receptors are localized on the membrane of endosomal compartments, where they interact with internalized CpG ODNs for signaling activation. [46][47][48][49] We therefore examined the subcellular localization of CpG FAM NP and CpG FAM NP@M R . LysoTracker is a fluorescent dye that labels acidic organelles such as the endolysosomes.
We monitored the ratios of CpG FAM NP and CpG FAM NP@M R that were colocalized with the LysoTracker signal. Although the uncoated and cell membrane-coated NP adopted different endocytic pathways, they both showed high and similar levels of endosomal localization (0.66 ± 0.04 vs. 0.74 ± 0.02), ensuring their interaction with TLR9 ( Figure 4A,B). were several fold higher than that in the CpG NP group, validating the high efficiency of CpG NP@M R for peripheral immunomodulation ( Figure 4C,D). The enhanced immunostimulatory effects might attribute to the faster cellular uptake efficiency of CpG NP@M R , as the equivalent amount of NP-bound cell membrane was unable to provoke the production of IL-6 or TNFα by itself ( Figure S6).
Microglia are tissue-resident macrophages in the central nervous system and are activated during brain infection, injury, or inflammation. [50][51][52][53] We further investigated the capacities of uncoated CpG

| DISCUSS ION
The potential applications of CpG ODNs are limited due to their susceptibility to biodegradation and poor efficiency in cell permeability.
Herein, a type of one-step self-assembled CpG NP was designed.
The CpG NP were further coated with the Raw264.7 macrophage cell membrane-or the BV-2 microglia cell membrane-derived vesicles for homotypic targeting to these two classes of immune cells.
Either type of membrane coating affected the physicochemical properties of CpG NP in a similar way and resulted in good biocompatibility. Besides, membrane coating led to accelerated rates in the cellular internalization of CpG NP@M R and CpG NP@M B . By our observation, the levels of internalized membrane-coated NP were at least one-fold higher than that of uncoated NP at various time points, confirming the feasibility of homologous membrane camouflage in the improvement of targeted delivery of CpG ODNs.
Accompanied with this observation, we found different endocytic pathways for uncoated and membrane-coated NP. While CpG NP were internalized predominantly via clathrin-mediated endocytosis, cellular uptake of membrane-coated CpG NP mainly relied on both caveolae-and micropinocytosis-mediated pathways.
After cellular internalization, the majority (~70%) of both uncoated and membrane-coated CpG NP trafficked to the endolysosomal compartments, allowing for their interaction with the TLR9 receptors. However, since the levels of internalized CpG NP were much lower than that of their membrane-coated counterparts, we observed higher levels of CpG NP@M R localized in the endolysosomes. Consistently with this finding, the membrane-camouflaged CpG NP induced higher levels of IL-6 and TNFα in macrophages and microglia, confirming their advantages in provoking immunostimulatory effects.
In conclusion, we designed a type of biomimetic NP, composed of self-assembled CpG NP core and cell membrane shell derived from macrophages or microglia, for the modulation of peripheral and central immunity. The utilization of cell membrane camouflage facilitated the targeted delivery of CpG NP to the cells with homologous membrane components, leading to more effective immune activation in peripheral and central immune cells. Our study thus provides a strategy for the application of CpG ODNs as a type of efficient and biocompatible immune adjuvant in disease treatment.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data supporting the findings of this study are available from the corresponding authors upon reasonable request.