To demonstrate the robust and predictable features of our selection strategy, we performed two distinct but identical selections against PC3 and LNCaP cells. They represent two distinct PCa epithelial cell lines that differ in their androgen responsiveness: androgen-responsive (LNCaP) and androgen-independent (PC3). RWPE-1 (prostate normal epithelial cell line), BPH-1 (prostate benign hyperplastic epithelial cell line), and PrEC (prostate normal epithelial cell line) have differential surface antigen expression as compared with LNCaP or PC3,23
and serve as model counter-selection cell lines to prevent the collection of RNAs that could bind to common surface antigens present on non-cancer cells. The starting RNA Apt candidate library was composed of 77 base long degradation-resistant RNA oligonucleotides incorporating 2'-OMe modified ATP, CTP, and UTP.24
The partly 2'-OMe-modified oligonucleotides were initially incubated with counter-selection cell lines (RWPE-1, BPH-1, and PrEC) consecutively and the RNA sequences remaining in the supernatant were continually collected. The collected RNAs were incubated with the target cells (either PC3 or LNCaP) at 37°C to allow for binding and cellular uptake. The cells were then extensively washed (rounds 1–12) and either lysed to collect the internalized RNAs (rounds 1–6), or treated with trypsin to remove the majority of membrane-bound RNAs prior to cell lysis and collection of internalized RNAs (rounds 7–12). The stringency of the selection was slowly increased by diminishing both the number of PC3 and LNCaP cells and the incubation time during the selection (rounds 1–12), and further increased by complicating the RNA pools through mutagenic PCR (round 7).25
The progress of selection, measured by the number of PCR cycles needed to amplify the chosen material for the next round, is shown in Figure S1
. As rounds of selection progressed, the needed PCR-cycle number steadily decreased from the 3rd
round, but did not decrease from 10th
up to 12th
round, thus indicating the saturation of Apt candidate enrichment.
Prior to identification of specific sequences in the round-12 RNA pool, we first confirmed that the enriched RNA pools (round 12 LNCaP and round 12 PC3), which represent many distinct Apt candidates, could be internalized and transported with NPs into target cancer cells. As a model NP platform, we used the hybrid lipid-polymer NP that has been designed and systematically investigated by our group.26–28
The hybrid NP consists of (i) a poly(D,L-lactide-co-glycolide) (PLGA) hydrophobic core for drug encapsulation, (ii) a lipid monolayer, and (iii) a poly(ethylene glycol) (PEG) shell. PEG was conjugated to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) at one end for interspersing into the lipid monolayer, and was functionalized with maleimide group at the other end for targeting ligand modification. This hybrid NP is prepared in a single-step process via
nanoprecipitation and self-assembly, and the yielded NP has the size of 50–100 nm and ζ potential of −10 to −20 mV, providing favourable physiochemical properties for drug delivery application. The conjugation of NP to RNA pool relies on maleimide-thoil chemistry (). Briefly, the vicinal hydroxyl groups in the unmodified 5'-end GTP of RNA pool were oxidized into aldehyde groups by periodate. These aldehyde groups further reacted with free amine group of cystamine to introduce thiol groups. The resulting thiolated RNA pools were then incubated with maleimide-functionalized NPs encapsulating NBD (22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol) to form NP (NBD)-RNA pool bioconjugates. As demonstrated in , the presence of the selected RNA round 12 LNCaP or PC3 pools greatly facilitated the uptake of the green fluorescent NPs into the target LNCaP or PC3 cells, separately. By contrast, control NPs similarly conjugated with the initial random library were not taken up by target cells at detectable levels. represents a panel of images across the Z-axis of a single cell with 3-D image deconvolution demonstrating the intracellular source of fluorescent signal, consistent with NP uptake within LNCaP or PC3 cells. The cell-uptake selection was shown to have successfully enriched a pool of Apt candidates that are specifically internalized by the cancer cells.
Figure 2 Demonstration of the internalization of selected round 12th RNAs-NP conjugates. A) RNAs and NP were conjugated by using maleimide-thiol chemistry. B) Cellular uptake of selected RNAs-NP conjugates. In all the images, the nucleus is in blue (DAPI), cytoskeleton (more ...)
We next separately cloned and sequenced the enriched PC3- and LNCaP-round 12 pools by using high-throughput genome sequencing methods. The sequences were sorted into putative families by aligning consensus motifs, and termed XEO1, XEO2, …, etc
. XEO2, XEO9 and their homologues represented 12% and 10% of the selected 68 sequences in the PC312th
round pool, separately. XEO6 and its homologues represented 14% of the selected 65 sequences in the LNCaP 12th
pool. These three abundant sequences, along with their truncated forms (XEO2mini and XEO6mini, described in supporting information, Figure S2
), were considered as the best internalizing Apt candidates for further characterization ().
Sequences of selected internalizing Apts.
We proceeded to characterize the internalization of the selected Apts. Because specific sequences had been identified, the synthesis, modification and labelling of Apts were directly performed by RNA synthesizers. This solid-phase chemical synthesis process is straightforward and accessible to be scaled up. Cy3-labeled Apts were incubated with target cells (PC3 or LNCaP) at 37°C for 2 h to allow for cellular uptake. Cells were then treated with trypsin to remove the external binding fluorescence signal that could interfere with the detection of the intracellular Apts,20, 29
followed by flow cytometry analysis. Cells were incubated with similarly synthesized Cy3-labeled initial RNA random library as a control and trypsinized to determine non-specific background uptake. shows the representative results from one of the selected Apts (XEO2). Compared with the initial library, the XEO2 profile showed a clear right shift in cytometric analysis, suggesting uptake by PC3 cells (). We further evaluated uptake of Cy3-labeled XEO2 during 2h incubation with various concentrations. The internalization of the selected XEO2 Apt was enhanced in a concentration-dependent fashion and reached a plateau in target PC3 cells (). By comparison, uptake of the initial library showed only a slight linear increase. The difference in the cellular uptake profiles indicates that, unlike the non-specific cellular uptake shown by random sequences, receptor-mediated endocytosis might participate in the specific and efficient cellular uptake of the selected XEO2 Apt.30–32
Confocal images further confirmed the cellular internalization of Cy3-labeled XEO2 ().
Figure 3 Internalization of Apt XEO2. A) Representative flow cytometric profiles showing XEO2 internalization signals in PC3 cells. The black curve represents the background uptake of unselected initial library. B) Uptake efficiency of XEO2 by PC3 Cy3-labeled (more ...)
Besides XEO2, the other selected sequences also exhibited cellular uptake into target cancer cells (, additional examples were shown in Figure S3, S4, S6
). Using R value as the criteria () to measure internalization capacity, we quantitatively compared selected Apts with a well-studied A10 Apt that bind to prostate-specific membrane antigen (PSMA). A10 gets taken up into PSMA-expressed cells such as LNCaP, but not PC3 cells that do not express PSMA antigens. As shown in Figure S7
, the R value of A10 in LNCaP cells was 1.45 (1<R<1.5, ++). As such, the internalization capacity of XEO2, XEO6, XEO6mini (XEO6 truncated form), and XEO9 (R>2, ++++, as summarized in ) was higher than that of A10 (1<R<1.5, ++) in LNCaP cells, indicating the robust feature of “cell-uptake selection” strategy. In addition, our strategy allows, for the first time, to discover a group of new internalizing Apts XEO2, XEO2mini, and XEO9, which can get taken up into PC3 cells with high internalization capacity (R>2, ++++, as summarized in ). To the best of our knowledge, no cancer antigens and targeting Apt ligands have currently been identified for PC3 cells.33
Our strategy has the advantage for enabling the design and engineering of ligand-targeted NPs without prior knowledge of target antigens.
Cellular uptake of selected aptamers by different cell lines.
To ascertain whether these Apts were binding to cell-surface membrane proteins, cells were pre-treated with proteases, including trypsin and proteinase-K, before incubation with Cy3-labeled Apts. For example, although XEO2 showed the binding affinity of 117 nM with PC3 cells (Figure S5
), it lost the binding characteristics against target cells after protease treatments (), indicating that its target molecules are most likely membrane proteins. Protease treatment assays similarly showed these selected Apts likely bound to membrane proteins (Figure S8
). Further characterization of the protein could lead to the discovery of novel PCa biomarkers.
Taken together, multiple internalizing Apts targeting the same cancer cells were generated from a single selection process. Using multiple Apts for development of NP-Apt conjugates may be most clinically useful, whereas conventional single antigen-targeted NP platforms may be confounded by the heterogeneous pattern of intra- and inter-tumoral antigen expression.34, 35
Such a group of internalizing Apts isolated from our designed selection can collectively interact with multiple antigens on cancer cells, and potentially be utilized to develop a multi-antigen targeted NP platform to address this limitation.
We subsequently assessed the cell-type specificity of selected internalizing Apts. As illustrated in , Apts XEO2 and XEO9 showed specific uptake into both LNCaP and PC3 cells. Apts XEO6 and XEO6mini showed specific uptake only into LNCaP cells. Apt XEO2mini showed specific uptake only into PC3 cells. All these five sequences showed much less favourable uptake into other cell lines, including BPH, RWPE-1, HeLa, SKBR3, A375, U373MG, T98G, U-87MG, A549, and SKOV-3. The slightly uptake into some of these cells lines may be due to the fact that some biomarkers, which are expressed in prostate cancer cells, are also expressed in non-prostate cancer cells albeit at a relatively lower expression level. For example, PSMA over-expressed in PCa cells, is also expressed at various degrees in normal prostate and other normal tissues, including whole brain, kidney, liver and small intestine,36
and is similarly over-expressed on the neovasculature of most non-prostate solid tumors.37, 38
The XEO2mini, XEO6, and XEO6mini had the most specific internalization profiles among the selected Apts, and thus may be promising for targeted delivery applications.
To investigate the feasibility of using the selected internalizing Apts for NP incorporation into potential applications, we used the XEO2mini as a representative Apt to develop a model system of NP-Apt bioconjugates. The conjugation of Apt XEO2mini and NP was achieved by using maleimide-thiol chemistry — the Apt was modified by solid-phase synthesis with a thiol group at its 5' end, and the NP was pre-functionalized with maleimide. We previously have demonstrated the optimal density of A10 Apt on the NP surface for in vitro
and in vivo
With the determined optimal density of one Apt per 1180 nm2
of NP surface area,39
we anticipate our NPs with a diameter of 80 nm have approximately the density of 17 Apts per NP. We visualized the cellular uptake of the NP-Apt XEO2mini (NP-Apt) by encapsulating NBD inside the NPs; though for clinical applications, small molecule drugs, siRNAs or other therapeutics may be encapsulated. PC3 and HeLa cells were employed as model target and non-target cell lines, respectively. As shown in , the cellular uptake of NP(NBD)-Apt was significantly enhanced in the target cells compared with that of the non-conjugated NP(NBD). The differential uptake of the NP(NBD)-Apt was not observed in the non-target cells. The background NBD signal represented nonspecific cellular uptake of NPs and any free NBD released from the NPs during incubation. The high magnification imaging () shows the cellular uptake and cytoplasmic distribution of the NP(NBD)-Apt inside the target cells. In addition, flow cytometry analysis was performed to confirm specific cellular uptake of the targeted NP-Apt (Figure S9
Figure 4 A) Representative confocal images showing specific uptake of NP-XEO2mini conjugates in different cells. NBD cholesterol is encapsulated in the NP. In each image, left panel: fluorescent image; right panel: overlay of fluorescence and optical image. Targeted (more ...)
With the model system of the XEO2mini-conjugated NPs, we next investigated its potential efficacy for drug delivery by encapsulating docetaxel (Dtxl) inside the NPs. A control experiment was first performed by incubating the cells with Apt XEO2mini or NPs without drug in both non-conjugated and Apt-conjugated forms. No obvious cytotoxicity was found in either target or non-target cell lines ( and Figure S10
), confirming the non-cytotoxicity of NPs and Apt XEO2mini. After loading with Dtxl, we observed the differential cytotoxicity of Dtxl-NP in non-target and target cells, which may be due to the differences in the non-specific uptake of NPs and in the IC50
of Dtxl between two cell lines.40–42
To exclude these intrinsic factors, we compared the cytotoxic effects of Dtxl-NP-Apt and Dtxl-NP in the same cell line, and thus each line is its own control. As shown in , the Dtxl-NP-Apt (71.45% ± 3.60) showed similar cytotoxicity to the Dtxl-NP (75.33% 2.21) in non-target cells (mean ± SD, n=5, P>0.05). In contrast, the Dtxl-NP-Apt (63.10% ± 5.81) was significantly more cytotoxic than the Dtxl-NP (85.47% ± 3.65) in target cells (mean ± SD, n=5, P<0.001). The significant increase in cellular cytotoxicity is presumably through Apt-targeted intracellular delivery and release of Dtxl in target cells. Previously, we had developed Dtxl-encapsulated and A10 Apt-targeted NP that bound to extracellular domain of the PSMA protein on the surface of PCa cells, and explored the efficacy of this system in vitro and in vivo
In that study, we showed an enhancement in the cytotoxicity of A10-conjugated Dtxl-NP-Apt (42% ± 2) compared with Dtxl-NP lacking the A10 Apt (61% ± 5).3
Our newly-developed internalizing NP-Apt system showed at least equivalent or more favorable enhancement in therapeutic efficacy than A10 Apt-targeted NP delivery system, demonstrating the potential of this system for targeted cancer therapy. More importantly, unlike the A10 targeted NPs which recognized the well characterized PSMA protein, the current platform allows us to develop equally efficacious or better targeted NPs even when the target antigen is unknown.