Conditional Cell-Penetrating Peptide Exposure as Selective Nanoparticle Uptake Signal

A major bottleneck diminishing the therapeutic efficacy of various drugs is that only small proportions of the administered dose reach the site of action. One promising approach to increase the drug amount in the target tissue is the delivery via nanoparticles (NPs) modified with ligands of cell surface receptors for the selective identification of target cells. However, since receptor binding can unintentionally trigger intracellular signaling cascades, our objective was to develop a receptor-independent way of NP uptake. Cell-penetrating peptides (CPPs) are an attractive tool since they allow efficient cell membrane crossing. So far, their applicability is severely limited as their uptake-promoting ability is nonspecific. Therefore, we aimed to achieve a conditional CPP-mediated NP internalization exclusively into target cells. We synthesized different CPP candidates and investigated their influence on nanoparticle stability, ζ-potential, and uptake characteristics in a core–shell nanoparticle system consisting of poly(lactid-co-glycolid) (PLGA) and poly(lactic acid)-poly(ethylene glycol) (PLA10kPEG2k) block copolymers with CPPs attached to the PEG part. We identified TAT47–57 (TAT) as the most promising candidate and subsequently combined the TAT-modified PLA10kPEG2k polymer with longer PLA10kPEG5k polymer chains, modified with the potent angiotensin-converting enzyme 2 (ACE2) inhibitor MLN-4760. While MLN-4760 enables selective target cell identification, the additional PEG length hides the CPP during a first unspecific cell contact. Only after the previous selective binding of MLN-4760 to ACE2, the established spatial proximity exposes the CPP, triggering cell uptake. We found an 18-fold uptake improvement in ACE2-positive cells compared to unmodified particles. In summary, our work paves the way for a conditional and thus highly selective receptor-independent nanoparticle uptake, which is beneficial in terms of avoiding side effects.


2.5
R10prot.Therefore, the coupled cell-penetrating peptide (CPP) was localized on the nanoparticle surface.

Mass spectrometry
Different mass-ratios of CPP-modified polymer and unfunctionalized, uncharged methoxy polymer were evaluated as indicated in the diagrams.By modifying the particles with CPPs, the size increased for all ligands compared to unmodified control nanoparticles (n=3 technical replicates).Nanoparticles were covalently core-labeled with Cy5 (red).Cell nuclei were stained with DAPI (blue).
After 1 h of incubation, the majority of the nanoparticles were bound to the nanoparticle surface.After an additional incubation period of 16 hours, the nanoparticles were taken up into the cells.Compared to particles with unshielded R10 a shift of cytotoxicity to higher nanoparticle concentrations could be detected.The cytotoxicity of MLN-TAT NPs and control NPs was additionally investigated with HEK293T-ACE2 cells under the conditions of the flow cytometry experiments.Since HEK293 cells show only weak adherence, the utilized 96-well plates were initially coated using Collagen A (Biochrom GmbH, Berlin, Germany).Therefore, 1.25 mL collagen A 1 mg/mL was mixed with 11.25 mL PBS pH 2.4 (pH adjusted with 37% hydrochloric acid), and 250 µL/well was added.The solution was incubated for 1 h at 37 °C.Afterwards, the collagen was removed carefully.The wells were washed using 250 µL DMEM+10% FBS.Directly after the coating step, 75,000 cells/well were seeded into the coated plate and incubated for 24 hours at 37 °C.
Unlabeled MLN-TAT NPs and control NPs were prepared according to chapter 2.4 and the particle concentration was adjusted to 100 pM with Leibovitz Medium.The cell medium was aspirated and the particles were added and incubated for one hour.Subsequently, the particle solution was aspirated and the cells were incubated with 200 µL MTT working reagent, prepared as described in chapter 2.6, for 3 hours at 37 °C.All the following steps were performed according to chapter 2.6.A cell viability of 99.86±5.48% was demonstrated after treatment with MLN-TAT-NPs and 92.97±6.51%after treatment with control-NPs.
Figure S29.CLSM images of time dependence of R7-modified NP uptake into HEK293 cells.(A) Procedure ibidi-slide preparation and Z-stack of nanoparticle binding/uptake after 1h of incubation time.(B) Procedure ibidi-slide preparation and Z-stack of nanoparticle binding/uptake after 1h of incubation and additional 16 hours of incubation after nanoparticle expiration and washing.

Figure S30 .
Figure S30.Shielding of R7 by PLA 10k PEG 5k COOH polymer led to the loss of zeta-potential and significant uptake improvement-correlation in HEK293 cells.The cells were incubated with Cy5labeled nanoparticles for 1 h at 37°C and afterwards analyzed via flow cytometry.(A) Zeta potential measurements of the particles.(B) Flow cytometric evaluation.Results represent mean ± SD (n = 3, levels of statistical significance are indicated as *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).

Figure S32 .
Figure S32.Effect of CPP shielding on R10-NP-cytotoxicity.The CPPs were sterically shielded by the usage of 25% longer polymers (PLA 10k PEG 5k COOH) and the cytotoxicity was evaluated via an MTT assay performed with L929-cells.The different DOMs investigated are indicated in the figure legend.

Figure S33 .
Figure S33.Evaluation of the cytotoxicity of NPs surface-modified with TAT(47-57).The MTT assay was performed with L929 cells.The different DOMs investigated are indicated in the figure legend.

Figure S 35 .
Figure S 35.Evaluation of the cell viability of HEK293T-ACE2 cells under flow cytometry experimental conditions.(n=6)