The impact of conjugation strategies and linker density on the performance of the Spermine-AcDex nanoparticle-splenocyte conjugate

Abstract

A common approach in living medicine engineering is modifying cell surfaces with nanomedicines to form nanoparticle-cell conjugates. Despite various available strategies, limited research has examined how conjugation strategies affect the efficiency and stability of the delivery systems. Herein, we prepared polymeric nanoparticles (NPs) with protein payloads and modified them with different linkers. These NPs were conjugated to primary splenocytes using either covalent or electrostatic interactions, followed by flow cytometry analysis to evaluate the conjugating efficiency and stability. The results demonstrated that electrostatic interactions were more effective in achieving conjugation, whereas covalent interactions provided greater stability. Furthermore, the linker density on the nanoparticle surface also affected the stability. After three days of in vitro culture, NPs with fewer linkers were predominantly internalized by the splenocytes, whereas those with more linkers partially remained on the cell surface. Overall, this study provides fundamental insights into nanoparticle-cell conjugation, thereby contributing to living medicine design and engineering for therapeutic applications.

Scheme 1. The scheme of NCC conjugation strategies via electrostatic or covalent interactions. Created using BioRender.com.

Fig. 1. The preparation and characteristics of BSA-loaded NPs. (A) The scheme of nanoparticle preparation. (B–D) Size, PDI, and fluorescence intensity in different media over 3 days. (E) The release behavior of NPs at different pH values. Mean ± SD (n = 3).

Fig. 2. The physiochemical properties of NPs modified with BMPS, DTSSP, DSS, and SPDP. (A) The scheme of NPs modified with BMPS (Mal-S), DTSSP (Amide-S), DSS (Amide), and (SPDP) Pyr-S. Results of Mal-S, Amide-S, Amide, and Pyr-S with a high or low degree of modification on (B) size, (C) PDI, and (D) zeta potential. (E) The cell viability of splenocytes incubated with different amounts of NPs. (F) The flow cytometry histograms and (G) the mean fluorescence intensity (MFI) of splenocytes conjugated with different amounts of NPs. Mean ± SD (n = 3). For C, data were analyzed by the one-way ANOVA test, * P < 0.05.

Fig. 3. The stability of NCCs in vitro. Fabrication and culturing of the NCCs and then quantitative analysis of the percentage of positive events (splenocytes modified with NPs) after (A) 0 day, (B) 1 day, and (C) 3 days in vitro and the MFI of NCCs after (D) 0 day, (E) 1 day, and (F) 3 days in vitro. Mean ± SD (n = 3). *p < 0.05, **p < 0.01 ***P< 0.001, one-way ANOVA with Tukey's HSD to determine significance between NPs and other groups.

Fig. 4. Investigation of the stability of NCCs via biotin–streptavidin interactions in vitro. (A) The scheme of probing biotinylated NCCs with AF488-streptavidin in vitro. (B) The scheme of different nanoparticle–cell association status in the flow cytometry results. Q1: NCCs with internalized NPs; Q2: NCCs with surface-conjugated NPs; Q3: NCCs with nonspecific streptavidin binding; and Q4: cells without NPs or streptavidin. (C)–(K) The flow cytometry results of different NCCs on day 0 (red dots) and day 3 (blue dots): (C) Pyr-S (High), (D) Amide (High), (E) Amide-S (High), (F) Mal-S (High), (G) NPs, (H) Pyr-S (Low), (I) Amide (Low), (J) Amide-S (Low) and (K) Mal-S (Low).

Fig. 5. Summary of the conjugation strategies. Evaluation of the NPs with a low degree of modification in terms of (A) conjugation efficiency index and (B) stability and NPs with a high degree of modification in terms of (C) conjugation efficiency index and (D) stability.