Enhanced cell target specificity and uptake of lipid nanoparticles using RNA aptamers and peptides

Lipid nanoparticles (LNPs) constitute a facile and scalable approach for delivery of payloads to human cells. LNPs are relatively immunologically inert and can be produced in a cost effective and scalable manner. However, targeting and delivery of LNPs across the blood brain barrier (BBB) has proven challenging. In an effort to target LNPs to particular cell types as well as generating LNPs that can cross the BBB, we developed and assessed two approaches whereby BBB penetrating peptides Tat or T7, and RNA aptamers targeted to gp160 from HIV or CCR5, a HIV-1 co-receptor, were incorporated into LNPs. We report here that a CCR5 selective RNA aptamer acts to facilitate entry through a simplified BBB model and to drive the uptake of LNPs


Introduction
Lipid nanoparticles (LNPs) represent an effective platform for delivering of small molecules, RNA or DNA into target cells [1]. LNPs have been successfully deployed via different administration routes in vivo to distribute their cargo into target tissues [2][3][4][5][6][7][8]. By changing lipid composition [6] and/or including short peptides [9] and ligands [10], one can modulate the biodistribution of the LNP in the body. However, despite these advances, targeting of LNPs to the brain tissue remains a challenge [11].
In order to reach safer therapeutic options for treatment of brain diseases and disorders, a productive drug transport across the blood-brain-barrier (BBB) is critical. For example, despite successful implementation of antiretroviral drugs for the treatment of human immunodeficiency virus (HIV-1), HIV-1 associated neurological disorders persist due to the poor uptake of antiretroviral drugs across the BBB [12][13][14]. There are two ways to traverse the BBB, one is through temporary disruption of the physical barrier, that impairs BBB function, the other is to use nanocarriers or particles [11]. The latter presents a non-invasive route that is safer than physical disruption [11]. One approach to increase transport of LNPs through the notoriously protective BBB is to use short positively charged peptides or receptor specific ligands, both of which have shown to be effective at increasing transport of LNPs, nucleotides and small molecules through the BBB [9,[15][16][17]. For example, the short positively charged peptide, Tat, has previously been demonstrated effective as an excipient species to increase the uptake through the negatively charged BBB [9,18]. Tat (H-YGRKKRRQRRR-NH2) is an arginine rich short cell penetrating peptide derived from the natural nuclear trans-activator of transcription (Tat) protein of HIV-1 [19,20]. The HIV-1 Tat protein itself, has been shown to traverse the BBB by acting as a cell-penetrating peptide [9,20]. Other small positively charged molecules used for BBB penetration is transferrin and its peptide derivatives or analogs that act as ligands for the transferrin receptor. The transferrin receptor is highly expressed in brain capillaries, nucleated cells and in rapidly dividing cells [21], and its endogenous ligand transferrin, has previously been used to increase transport of small molecules and oligonucleotides across the BBB [21][22][23]. The seven-amino-acid peptide T7 (H-HAIYPRH-NH2) was identified via phage display [24] and has a high affinity (~10 nM) for the transferrin receptor [24,25]. This peptide does not compete with endogenous transferrin binding and has been used to successfully enhance the drug delivery to brain tissue [15,22,[24][25][26]. Both peptides were included in this study and modified with an N-terminal lipid anchor for LNP post-insertion. The design of the lipid anchor includes two palmitoyl chains that are attached through a 1,2-diamino propanoic acid (Dap) moiety on the N-terminus of each peptide, providing the lipopeptides dipalmitoyl-Dap-T7 and dipalmitoyl-Dap-Tat. Double lipidation ensures a more stable lipid membrane anchoring compared to a single fatty acid chain or cholesteryl variant [27][28][29]. The careful choice of Dap and palmitic acid allows for the entire synthesis to be performed on solid support with no need for additional reactions after cleavage [27][28][29].
One approach to generate LNP formulations with higher specificity for antigen-expressing cells is to use RNA aptamers. RNA aptamers are short oligonucleotides that are evolved using a process called Systematic Evolution of Ligands by Exponential Enrichment (SELEX) [30].
SELEX is an iterative process that begins with a large oligonucleotide library which, through a process of negative and positive selection, ends with a few candidates that are specific for a particular protein [30,31]. Using HIV-1 as our model, we explored the use of two RNA aptamers as a mean to increase the specificity of LNPs for HIV-1 infected and/or target cells [31]. RNA aptamers are ideal candidates due to their lower immunogenicity profile than their DNA counterparts [30,32,33]. RNA aptamers are also highly amenable to forming complex and dynamic secondary structures that makes them ideal molecules for novel ligand development [31]. Zhou et al., previously reported on an RNA aptamer specific for the HIV-1 entry co-receptor C-C chemokine receptor type 5 (CCR5) [34] and an RNA aptamer specific for the HIV-1 envelope protein gp160 [35]. The CCR5 RNA aptamer G-3, has been shown to be specific for and internalized by the CCR5 receptor [34]. Similarly, the A-1 aptamer has been found to specifically recognize gp160 and that it may be internalized through receptor-mediated endocytosis [35].
Thus, both aptamers present an additional potential route for LNP internalization and target cell specificity. In order to assess the ability of aptamers to drive LNP internalization, short complementary Cy5-DNA oligonucleotides specific for each aptamer were used as probes to detect LNP uptake in different cells.
In this study, we employed lipid compositions and formulation procedures previously reported in literature [4]. Specifically, the cationic and ionizable DLin-MC3-DMA lipid is a constituent of the FDA-approved LNP-formulated small interfering RNA (siRNA) drug Patisiran ® for treatment of familial transthyretin amyloidosis [36,37]. Clinical trial safety assessments of this formulation showed no liver toxicity and no immune stimulation with ~10% of trial participants experiencing mild to moderate adverse events upon administration [38]. It includes encapsulation of siRNA by a mixture of lipid components such as an ionizable cationic lipid, distearolyphosphatidycholine (DSPC), cholesterol and PEG-lipid, each with an essential role in the design. These lipids promote the effective distribution of the LNP in vivo as well as aide in effective cargo release from the endosome [1,37].
To this end, we herein report the efficacy, delivery capability and functionality of the addition of peptides and RNA aptamers in facilitating entry through a simplified BBB model as well as to determine whether inclusion of these molecules could facilitate cell specific uptake. We further show that LNPs generally exhibit a low immunogenic and toxic profile and that RNA aptamers can act as potential enhancers to effectuate the delivery of LNPs into the CNS.

Lipid nanoparticle development and characteristics
In accordance with a previously published procedure, we generated LNPs using a mixture of DLin-MC3-DMA, DSPC, cholesterol and DMG-PEG 2000. Lipids were first extruded then complexed with negatively charged aptamers annealed with fluorescently tagged complementary DNA oligonucleotides (GP160:A-1 or CCR5:G-3) to simultaneously assemble the LNPs. At this stage, the LNPs were examined by DLS ( Table 1). While non-complexed (empty) LNPs had an average size of 62.4 nm and zeta potential (ZP) of -2.9 mV, LNPs mixed with GP160:A-1 and CCR5:G-3 displayed average sizes of 57.3 nm and 91.9 nm, and more negative ZPs (-11 mV and -9.4 mV), respectively ( Table 1). These ZP values indicate that complexation leads to a neutral to anionic LNP product [39], properties that typically confer with low to no cytotoxicity in vivo [40]. Further, the additional decrease in the ZP indicates efficient aptamer loading into the LNPs. Additionally, low polydispersity index (PDI) values is reported for both formulations (Table 1) indicating a relative high degree of monodispersity.
Sample list: Listed samples used in present study.

LNP sample
Cy5 DNA probe:aptamer Tat   LNP B9 G-3 T7  G-3:CCR5  T7 LNP B9 G-3 Tat G-3:CCR5 Tat  Figure S2). After post-insertion, LNP sizes were found by NTA to range from 54-66 nm ( Table 2), while TEM analysis revealed average sizes between 45-52 nm (Supplementary Figure 2B). While there appears to be a ~10 nm discrepancy when comparing DLS and NTA with TEM, this size difference was found to be consistent between method of analysis for all sample.
For example, LNP B9 T7 was characterized by the smallest average size using both NTA (~54 nm) and TEM (~45 nm). Thus, the average sizes obtained by NTA are in agreement with the average size observed using TEM (Supplementary Figure 2A and Table 2). Similarly, while the mean diameter of LNP B9 G-3 was found to be larger by DLS (91.1 nm) than the values reported for NTA (67.2 nm) and TEM (52 nm), the sizes of the LNP B9 and LNP B9 A-1 samples via DLS are also in agreement with the NTA and TEM reported sizes. These discrepancies may be indicative of the inherent differences between these three analysis methods and highlight the need to confirm LNP sizes using more than one technique. Nevertheless, the small size of these nanoparticles (<100 nm) is ideal for in vivo applications as they may bypass the reticuloendothelial system and thereby increase LNP circulation time in vivo [41].

LNPs with post-insertion T7 peptide
Previous studies have demonstrated the ability of the T7 peptide to increase LNP transport across the blood brain barrier (BBB) [22][23][24]42]. In order to test this, we used a simple transwell assay with human brain endothelial cells (hCMEC/D3) that were cultured on a 0.4 µM transwell mesh until a trans-endothelial electrical resistance of above 30 .cm 2 was reached. This measure is an indicator that a tight junction barrier has formed within these cells and can be used to determine the ability of the LNPs to pass through the BBB (Supplementary Figure 3A).
Additionally, we further confirmed our junctions using fluorescent microscopy on the barrier layers to confirm expression of claudin-5, a known tight junction protein (Supplementary Figure   3B). We observed that LNPs were readily taken up by both HeLa and TZM-bls in the absence of a transwell insert (Figure 1A-B). With the addition of the hCMEC/D3 cells in the apical chamber, we found that HeLa cells were less Cy5 positive (~60%) than the target TZM-bl cells (~100%) ( Figure 1A). Further, when examining the intensity of Cy5 in these cell populations, we found that the addition of the T7 peptide increases uptake by 1.2 fold through the hCMEC/D3 cellular barrier, while also increasing uptake through direct addition by 1.6 -1.8 fold ( Figure 1B).
Additionally, the mean fluorescent intensity (MFI) was found to be 2-2. In contrast, we found that formulating LNPs with the gp160 specific A-1 aptamer did not result in any significant increase in percentage uptake in the target gp160 positive HEK293T cells compared to HEK293T cells alone ( Figure 1C). However, we did observe that the MFI in gp160 positive HEK293T to be 1.3 and 1.45 fold (barrier, and non-barrier groups respectively) higher than in the HEK293T cells alone (Figure 1D), suggesting higher levels of LNPs in gp160 expressing HEK293T cells. We also observed that direct addition of the LNPs resulted in a higher percentage of Cy5 positive cell detection and a higher MFI compared to the hCMEC/D3 barrier ( Figure 1D, Supplementary Figure 4).
Collectively, these data suggest that the candidate LNPs, particularly the LNP B9 G-3 T7, may increase uptake through tight junctions and prove useful in transiting drugs and small cargo through the BBB in vivo.

LNPs with post-inserted Tat peptide
Tat is a cationic peptide that is known to increase transport of molecules through the blood brain barrier and increase uptake into cells [18]. In a similar manner to the transferrin peptide (T7), we investigated the ability of Tat to drive LNP uptake in cell lines. Interestingly, we found that the addition of the Tat peptide to either the A-1 or G-3 complexed LNPs did not have any effect on BBB penetration (Figure 2 A-D). Rather, we observed that LNPs containing the G-3 aptamer showed an increase uptake in target cells expressing CCR5 (Figure 2 A-B). We observed that TZM-bls had a ~98% uptake of LNPs via the hCMEC/D3 barrier compared to ~63% in HeLa cells ( Collectively, these data suggest that the addition of Tat to LNPs have no effects on BBB transit when compared to the T7 peptide. We further found that A-1 aptamer incorporation into the LNP formulation does not appear to enhance specific targeting of gp160 expressing cells either through the hCMEC/D3 barrier or through direct addition, suggesting that it may not be an ideal candidate moving forward.

LNPs do not stimulate an immune response
In order to further characterize LNPs we decided to evaluate their immunogenic profile. We stimulated monocytes obtained from whole blood for 6 days with 10 ng/mL granulocytemacrophage colony-stimulating factor (GMCSF). This programs the monocytes to form macrophages that are primed to respond in a type 1 manner. After 24 hrs of stimulation with either the LNPs or positive controls for an RNA/DNA response (poly I:C) or a bacterial response (LPS), we found that the LNPs did not increase secretion of any of the cytokines tested (IL-1β, IL-10, IL-6, IFN-ɣ, TNFα, IL-2, IL-4, IL-8 and IL-5) above basal (PBS) conditions ( Figure 3A).
Additionally, we confirmed LNP uptake by the monocyte-derived macrophages (MDMs) using fluorescent microscopy ( Figure 3B). We found that all LNPs containing the Cy5 oligonucleotide were observable under the microscope (Figure 3B), and that all macrophages were 100% positive for Cy5. Additionally, using QuPath analysis software we determined the Cy5 MFI for each image. Interestingly, we found that the LNP A-1 and the LNP G-3 had higher MFIs in all the donors assessed compared to their Tat and T7 counterparts ( Figure 3C). Further, we found that the LNP G-3 exhibited the highest uptake in all the donors assessed ( Figure 3C). These 14 observations suggest that the candidate LNPs are relatively immunologically inert and may prove to be well-tolerated in vivo.

Aptamer and peptide LNPs have modest effects on cell viability in a cell specific manner
We next assessed whether LNPs could affect cell viability in HeLa and HEK293Ts. Cells were treated with the LNPs for 24 hr prior to performing the alamarBlue viability assay. In HeLa cells, we found that the LNP B9 alone had no effect on cell viability compared to the PBS control ( Figure 4A). Interestingly, we observed that cell viability was reduced by ~20% in HeLa cells treated with LNPs containing either A-1 or G-3 aptamer or LNPs with the Tat or T7 peptide alone ( Figure 4A). However, LNPs containing both the aptamer and a peptide (Tat or T7) did not further affect cell viability ( Figure 4A). This suggests that the aptamer and the peptides may contribute towards the loss of cell viability observed in this cell type. Conversely, we observed no loss of cell viability in HEK293T cells treated with LNPs containing either A-1 or G-3 aptamer, Tat or T7 alone, or the combination of aptamers and peptides ( Figure 4B). Like HeLa cells, the LNP formulation alone had no effect on cell viability ( Figure 4B). These data suggest that there may be some cell specific sensitivity toward the LNPs formulations, and that further studies are required to determine the optimal concentrations of aptamers and peptides within the LNPs, or to optimize the ratio of LNPs to cells in order to reduce toxicity in any cell line tested.

Discussion
LNPs represent an increasingly popular modality for cargo delivery. The vast improvements in lipid design and architecture have resulted in several successful LNP-driven vaccines and therapeutics including two RNA-based SARS-CoV-2 vaccines [43], as well as an siRNA-LNP for the treatment of a transthyretin amyloidosis [36]. However, further improvements in toxicity profiles, cargo-delivery and cell or organ specificity are needed to expand the use of LNPs for gene and drug delivery.
LNPs and aptamers have previously been used with great success to increase cell specificity. aptamers [46]. Taken together, work reported by these authors, as well as others, demonstrates usefulness of aptamers to increase cellular specificity and uptake of LNPs into the target cells.
In the present study, we observed that LNPs containing the G-3 aptamer targeting CCR5 resulted in a 40% increase in cellular uptake through the BBB and into target cells, and that these cells had higher LNP uptake (measured by a higher MFI) than their non-antigen expressing counterparts; while the gp160 aptamer (A-1) had no apparent effect on target cell uptake. One could speculate that this may be the result of the nature of the target proteins. CCR5, a cell surface receptor, is internalized upon ligand binding, before recycling back to the cell surface or processed for degradation in the lysosome [34]. On the other hand, gp120 is a viral surface protein that is involved in viral entry through complexation with CD4 and CCR5 or CXCR4 host cell surface receptors [35]. As such, gp160 expression on the host cell surface receptor, may not be as adept at facilitating cell entry via receptor-mediated endocytosis. Although Zhou et al., , observed by confocal microscopy that the A-1 aptamer entered gp160-positive cells and suggested that receptor-mediated endocytosis could be mechanism of entry, such a notion was not definitively demonstrated as the mechanism of uptake [35]. In addition, observed differences between these aptamers could also be due to differences in target receptor expression in the cell types, and/or differences in the affinity and specificity of these aptamers for their target receptors and/or differences in their mechanisms of uptake. Finally, the formulation procedure also likely influences the ability of the aptamers to act as productive ligands for their respective receptors, although more studies will be needed to fully delineate these effects.
One important aspect we set out to address was to identify proxies for successful LNP-mediated cargo delivery through the BBB and into the brain. As previously stated, effective transport systems for brain drug delivery are highly warranted. Herein, we find that the LNP platform can be applied as a vehicle to circumvent the BBB and effectively deliver oligonucleotide probes to antigen-expressing cell lines. In the case of HIV-1, there is currently a need for more effective delivery platforms compatible with anti-retroviral drugs. Specifically, a productive CNS delivery of such compounds is expected to reduce HIV-1 associated neurological disorders as well as to reduce HIV-1 replication at this sanctuary site [13,47,48].
We investigated the use of T7 and Tat peptides and evaluated their ability to aid delivery of LNPaptamer species across the BBB. We found that LNPs with either T7 or the Tat peptide did not significantly increase cellular uptake through the BBB above the LNPs containing aptamers alone. T7 appeared to have an effect on cellular uptake when the LNPs were directly added to the cells, and a small effect when applied through the apical chamber of the hCMEC/D3 cell line, while Tat had no effect. It may be prudent to dose the amount of post-inserted Tat or T7 peptide and in vivo [49,50]. In the work presented here, we immobilized Tat or transferrin onto the LNP formulations using a post-insertional technique. It could be that it would be more prudent to make the LNP formulation with the addition of Tat and T7 peptide during the initial synthesis.
Further, it may be important to increase the amount of post-insertional Tat and T7 used in future experiments, considering the concentrations we used were relatively low (~0.1% post addition).
Another approach is to use next generation of short peptides that also bind to the transferrin receptor at non-competing regions to endogenous transferrin in vivo [51]. These molecules are known as cystine dense peptides (CDPs), and have been shown to bind to the transferrin receptor in the picomolar range to facilitate BBB crossing in mouse models [51]. These short peptides may be advantageous to use when approaching an in vivo strategy especially considering that the concentration of the peptide needed in the formulation may be lower compared to the T7 peptide used in this study; however, its safety profile must still be fully evaluated.
Nevertheless, our LNPs, particularly the ones containing the G-3 aptamer alone resulted in BBB transport ranging from 50-65% in non-target cell lines, to 80-100% uptake in target cell lines.
Importantly, the hCMEC/D3 model represents a simplified representation of the BBB, which does not account for the full complexities of the BBB in vivo [52,53]. One could perform more complex in vitro assays that include a multicellular reconstruction of the BBB to also include astrocytes and microglial cells [54,55]. However, it may be more effective to perform further studies in nonprimate animal models to determine the efficacy of these LNPs in passing through the BBB.
Assessing and quantitating the percentage of LNP B9 to traverse the BBB is a critical step to determining its use as an effective LNP able to deliver small molecules or oligonucleotides into the brain. One important caveat to note is that the aptamers are species specific, thus the use of a xenograft model with human cells in a non-primate animal models are needed to determine the specificity of the LNP-aptamer tested.
Furthermore, while the LNP B9 alone had no effect on cellular viability, it appeared that the LNPs containing either the A-1 or G-3 aptamers, or the peptides, reduced cellular viability in HeLa cells by 20%, suggesting that there may be some toxicity when delivered to cells. However, these effects were not observed in HEK293T cells. It could be that the HeLa cell line is more sensitive than the HEK293T cell line. Nevertheless, the data suggest that further testing is required to determine the safety profile of these LNP aptamer and or peptide formulations. One way we could reduce the toxicity profile, is to chemically modify the RNA aptamers [33,56,57,58], or by reducing the aptamer concentration per LNP to thereby alleviate some of the observed the cellular toxicity. It could be that the RNA aptamer itself could contribute towards cell death, possibly through stimulating the retinoic acid-inducible gene 1 (RIG-1) pathway, and it may thus be prudent to assess IFN-α and IFN-β in the future [32,59]. Importantly, the LNP B9 formulation alone had no effect on cell viability, suggesting that the ratio of cationic and ionizable lipids is optimal and does not present acute toxicity issues. However, more work is needed to assess its toxicity in vivo, and in particular evaluate its effect on the liver [60]. Importantly, LNPs reported herein did not appear to stimulate an immune response in primary human monocyte-derived macrophages. Further, the addition of the aptamers and or the peptides in the LNP formulations had no effect on immune stimulation, suggesting that these LNPs and their modifications may be well tolerated in vivo. Importantly, both IL-6 and IFN-ɣ cytokines were not stimulated after exposure to the LNPs, suggesting that this LNP formulation may not induce cytokine release syndrome in vivo [60,61].
Taken together, we have shown that the LNP B9 formulation is safe, can traverse the BBB and is readily taken up in multiple cell types. In the future it will be interesting to explore whether increased uptake may also lead to increased delivery of target molecules such as siRNA, mRNA

RNA and DNA oligonucleotides
The RNA aptamers and Cy5 DNA oligonucleotides were synthesized at the RNA/DNA synthesis core at City of Hope (Duarte, CA). The RNA aptamers, A-1 [35] and G-3 [34] were developed by This procedure was repeated two more times, followed by a lyophilization step to receive the crude peptide. The crude T7-lipid conjugate was purified by normal phase chromatography utilizing gradient elution (2-50% MeOH in DCM). The desired modified T7 peptide was characterized via MALDI-TOF spectrometry (Bruker, MA) (Supplementary Figure 1B) and isolated as a colorless powder (9 mg, 6 mol, 6% yield

Cell lines and maintenance
HeLa and HEK293T were purchased from American Type Culture Collection (ATCC, VA). TZMbls were acquired through the NIH AIDS reagent program and were engineered to express high levels of the HIV-1 co-receptor CCR5 [62]. HEK293T-gp160 cells were a kind gift from Dr. Bing Chen (Harvard, MA), and stably express the 92UG037.8 strain of the viral envelope protein, Env [63]. The human brain endothelial cell line hCMEC/D3 was purchased from Millipore Sigma

Inflammation assay
Blood from consented and de-identified donors was used in this study under an approved IRB