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Nano and Micro Technologies

Nano and Micro Technologies - Poster Session B

Poster J13 - Designing cell-penetrating peptide-conjugated nanoparticles for enhanced delivery through intestinal mucosa

Thursday, October 24, 2024
2:45 PM - 3:45 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Oral delivery systems benefit from ease of administration, though the efficacy of this route is often hampered by enzymatic degradation and poor drug solubility. Loading drugs into nanocarriers can address these issues. However, the intestinal mucosa also presents a significant barrier to reaching systemic circulation: large and charged carriers will become trapped within the matrix of gel-forming mucins in the small intestine, reducing uptake into underlying epithelial cells. Grafting small nanocarriers with neutrally charged polyethylene glycol (PEG), a hydrophilic polymer that can increase the circulation time of conjugated carriers and reduce mucoadhesion, can improve mucus penetration and allow nanocarriers to reach cells.
Cell-penetrating peptides (CPPs), short sequences that promote direct penetration through the cell membrane or endocytosis, can be conjugated to nanocarriers to enhance cell uptake and therapeutic efficacy. Some of the most commonly used CPPs, such as TAT, are cationic and enter cells through electrostatic interactions with the cell membrane, which presents a problem for mucosal delivery as the increase in charge post-conjugation can cause mucoadhesion. Thus, a CPP-conjugated and PEGylated nanoparticle system that is mucus-penetrating and cell-penetrating may be promising for oral delivery.
Most studies investigating CPP-conjugated nanocarriers do not focus on rational design; quantification of surface components and their relationships with nanoparticle size and charge and mucus penetration are not a primary concern. Here, we aimed to determine how CPP modification changes the surface characteristics of nanoparticles, if neutralization of surface charges is needed post-conjugation, and if CPP-modified, PEGylated nanoparticles demonstrate mucus diffusivity comparable to PEGylated nanoparticles.

Materials and

Methods:  We grafted methoxy-terminated (mPEG) and maleimide-terminated (malPEG) 5 kDa PEG-amine to carboxylated 100 nm red FluoSpheres (ThermoFisher) through EDC/NHS chemistry. PEGylated nanoparticles were then conjugated to cysteine-terminated TAT in 1x PBS through maleimide-thiol chemistry. We characterized nanoparticles before and after conjugation through dynamic light scattering (DLS) and phase analysis light scattering (PALS) in .1x PBS to determine how conjugation increased particle sizes and surface charges. We then developed a quantitative Fourier-transform infrared spectroscopy (FTIR) method to calculate PEG grafting based on the integral of the PEG ether peak by plotting peak areas measured for unconjugated mPEG and microsphere solutions against known mPEG concentrations. PEG density was determined based on Rf/D where Rf = aN3/5 (a is the length of a PEG monomer and N is the number of monomers in the chain) and D = 2A where A is the inverse of the PEG grafting density. The percentages of malPEG were determined using a modified version of the protocol from the Amplite Colorimetric Maleimide Assay Kit based on a standard curve of unconjugated malPEG and microsphere solutions. Lastly, we measured the diffusivity of the nanoparticles through 2% porcine gastric mucin and 4-arm-PEG-thiol hydrogels incubated on glass slides using MATLAB-based multiple particle tracking analysis and microrheology calculations. For each batch, 3 slides were prepared, and 3 4-second videos were captured at different positions within the gels. The first second of each video was used to determine the average diffusivity of the particles in each gel.

Results, Conclusions, and Discussions: We found that the hydrophilic diameters of PEGylated nanoparticles with CPP increased by ~ 5% (Figure 1A) compared to particles with PEG only, confirming that modification with the ~1.6 kDa TAT peptide slightly increased their sizes without inducing clumping. On average, the zeta potentials of CPP-conjugated and PEGylated nanoparticles were significantly higher than nanoparticles with the same PEG coat but no conjugated TAT and approached zero (Figure 1B). Using FTIR, we calculated Rf/D values and confirmed the presence of dense brush PEG conformations (Rf/D > 3) on all PEGylated batches (Figure 1C), which corroborates the near-neutral zeta potentials measured for all samples. Maleimide content on nanoparticles without peptide averaged at ~11%, and in most cases, the amount of free malPEG on nanoparticles with TAT was undetectable (Figure 1D). No significant differences in mucus diffusivity were observed and the variability within formulations was high, but the mean diffusivities suggest the following trend in penetration: 10% TAT-malPEG > PS-mPEG > 10% malPEG ~ PS (Figure 2).
We determined that conjugating cationic peptides to nanoparticles with a theoretical PEG grafting of 10% malPEG/90% mPEG increased surface charge to near neutral. Thus, conjugating anionic PEG to counter the increase in charge and synthesize near-neutrally charged particles is not required. Nanoparticle sizes slightly increased post-conjugation but remained within the range for efficient enterocyte uptake ( < 200 nm). Combined with the observed zeta potentials and trend in mucus diffusivity, these data suggest that CPP conjugation may enhance gut transport by neutralizing surface charges more effectively than PEGylation alone while maintaining a similar size. Future work will determine if conjugating anionic PEG to CPP-conjugated nanoparticles to balance the positively charged peptides on near-neutral nanoparticles will reduce interactions with mucus. Additionally, the sensitivity of the maleimide assay will be assessed to determine the minimum peptide conjugation efficiency measurable with this assay. If necessary, alternative methods that more accurately determine peptide grafting will be explored to ensure minimal free maleimide is available to bind to mucins. Further tracking experiments will also be conducted to confirm the observed trend in diffusivity.

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