Efficient delivery of plasmid DNA using incorporated nucleotides for

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Efficient delivery of plasmid DNA using incorporated nucleotides for precise conjugation of targeted nanoparticles Nathan Beals, Nithya Kasibhatla, and Soumitra Basu ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00596 • Publication Date (Web): 16 Jan 2019 Downloaded from http://pubs.acs.org on January 20, 2019

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ACS Applied Bio Materials

Efficient delivery of plasmid DNA using incorporated nucleotides for precise conjugation of targeted nanoparticles

Authors: Nathan Beals, Nithya Kasibhatla, and Soumitra Basu*

AUTHOR ADDRESS

Kent State University, Department of Chemistry and Biochemistry, Kent, OH 44242 KEYWORDS Nanoparticle, Drug Delivery, Plasmid DNA, Gene therapy, Cancer

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Abstract Many obstacles restricted development of DNA plasmid-based therapeutic delivery, involving but not limited to poor cellular uptake, premature material dissociation, and inefficient response. Additionally, lack of precision loading of the plasmids on the carrier nanoparticle may affect the overall nonspecificity in terms of loading as well as the site of loading. Here we report a strategy using the incorporation of a biotin-modified nucleotide into a 4.7 kb plasmid sequence for the site-specific nanoparticle conjugation as an improvement on targeted DNA plasmid delivery. Initially, a designed 80-nucleotide sequence was elongated by incorporating biotin-16aminoallyl-2'-dCTP that facilitated streptavidin binding as determined via polyacrylamide gel electrophoresis (PAGE). This modified sequence was ligated into a specific location of the EGFP plasmid to avoid possible interference with important functional elements and gene expression off of the plasmid. In parallel, a gold nanoparticle complex comprising of either a CD44 or mutant DNA conjugated aptamer, a PEGylated streptavidin, and a derivatized hyaluronic acid stabilizing polymer was synthesized. To delineate the ability of this nanoparticle-plasmid complex to exhibit an improved cellular delivery, MDA-MB-231 cells were treated with a set of plasmid and plasmid-nanoparticle complexes. Successful expression of EGFP was only observed in cells treated with the biotin-modified EGFP plasmid and a streptavidin-CD44 aptamernanoparticle. This demonstrated the need for the specific biotin-streptavidin binding to avoid nanoparticle-plasmid dissociation for improved efficacy. This proof-of-principle concept creates a flexible scaffold that can be assimilated into any plasmid that can produce small RNAs or encoding a therapeutic gene via an installation of a design that uses incorporated modified nucleotides as tethering points for nanoparticles which can play host to stabilizing ligands,


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additional therapeutic molecules and antibody conjugates among other possibilities. In our system, the nanoparticles are vehicles for the addition of targeting ligands that were essential for cell specificity and enhanced cellular uptake.



Introduction Nucleic acid therapeutics are unique compared to other drugs as not only do they target the most innate cellular processes involved within the central dogma but do it using existing cellular machinery. Human cells maintain diversity and function by using a sequence of events that can be oversimplified as genetic information of our DNA is transcribed into messenger RNA (mRNA) which is translated into proteins 1. Gene expression is a highly regulated process that is dependent on the needs of cells. One way to control this process is by using naturally occurring RNA interference (RNAi) pathways (siRNA, miRNA, and piRNA) which have been established to regulate the number of mRNA copies available for protein translation 2, 3. Another strategy of using DNA plasmids also takes advantage of the naturally occurring cellular machinery to produce various RNA products for modulating protein production 4.

One specific type of nucleic acid therapeutic molecule is non-viral DNA vectors, which have been shown to possess huge therapeutic advantages centered around gene therapy. Genetic mutations can be remedied by introducing a DNA plasmid to encode for a therapeutic gene that has previously been shown to inhibit expression or express small RNAi that can inhibit a gene that is overexpressed 5. Plasmid DNA encoding therapeutic proteins have been used in clinical trials, using non-viral and viral vectors for cellular delivery 6. However, both methods of delivery suffer from multiple disadvantages. Recent advancements have highlighted the possibility of encoding a Cas9 enzyme from a CRISPR plasmid that can specifically target genetic regions, via a guide RNA, that produces a double-stranded break

7, 8.

This double-stranded break can silence target

genes or engineer a specific change, generating the possibility of correcting diseased based


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mutations 9. In spite of various advantages that the nucleic acid based/targeted therapeutics possess, there are many obstacles that stand in the way of it becoming a reliable in vivo therapy. Non-viral vectors have very high molecular weights and carry a large negative charge due to the phosphate backbone, resulting in poor cellular entry, toxic side effects as well as having short lifetimes in biological systems due to degradation by nucleases

10.

Non-viral vectors are often

associated with non-specific toxicity as well 11. Also, delivering plasmids in a targeted site-specific fashion is still challenging. These have led to an increased interest in nanoparticle-plasmid DNA systems that try to circumvent pitfalls associated with plasmid DNA delivery. Lipid and polymer nanoparticles have been the most extensively studied systems thus far, utilizing cationic groups to bury and condense DNA plasmids for protection from nuclease degradation and neutralization of negative charges 4, 12. Previously, cationic polymers such as the naturally occurring chitosan have been seen to reduce the nonspecific toxicity of the non-viral delivery systems, while maintaining available functional groups to retain the possibility of additional loading of chemotherapeutics to enhance therapeutic index 13-15. Liposomes, which are made up of mixtures of lipids are another vehicle for DNA plasmid delivery but often suffer from inherent toxicity 16. However, they have shown improvements both in reducing DNA plasmid treatment toxicity and increasing cellular uptake by diversifying the liposome composition, specifically conjugating targeting aptamers on the liposome surface

17, 18.

But these common strategies can have size limitations that can

potentially result in a loss of passive targeting through the enhanced permeability and retention (EPR) effect that is inherent to tumor models. Nanoparticles of 100 nm in size are deemed to be most effective for the EPR based localization, but larger sizes, for example, ones nearing 400 nm cannot pass through the gap junctions of the capillary cell lining 19. Gold nanoparticles (AuNP) have displayed the ability to protect DNA plasmids from nuclease activity through conjugated



PAMAM (polyamidoamine) polymers to the gold surface that create charge interactions with the negative phosphate backbone

20, 21.

This AuNP-PAMAM polymer coating has been shown to

condense and protect plasmids, but potential problems exist because of uncontrolled interactions between the DNA backbone and AuNP-PAMAM complex. The interaction of polycationic polymers and plasmid backbone based on charge-charge interactions can preclude the DNA vector from being active to its full potential without the full dissociation of the complex prior to cellular entry or within cell 22.

In an effort to avoid some of the pitfalls mentioned earlier and improve upon plasmid nanoparticle drug delivery systems, a number of reports within the literature observe how non-viral delivery vectors have been made to specifically release the plasmid vector from the inherent polymer complex. Stimuli based complexes have been created to overcome non-specific interaction of the nano-material to the plasmid vector but in a number of situations, only a minimal amount plasmid is released from the complex 23. We decided to create a strategy that circumvented this problem of non-specific polymer-DNA binding in an effort to create a more precise and controllable non-viral vector delivery system. In this report, modified nucleotides were incorporated into specific sites of the plasmid, creating specific conjugation points that could be used for nanoparticle attachment. The nanoparticles would then be a platform for a multitude of interactions of various polymers, specifically targeting aptamers for cellular uptake. The pre-planned location of modified nucleotides (biotin modified) was created to avoid interaction and interference within the regions important for plasmid function. We characterized this new nanoparticle-plasmid delivery system, then delivered a biotin incorporated EGFP plasmid to CD44 overexpressing triple-negative breast cancer cells (TNBC). Confocal microscopy and EGFP mRNA measurements established this


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novel delivery system can be uptaken in targeted cells and undergo EGFP expression. We believe this can be a highly controllable modular nanoparticle complex for cellular delivery of therapeutic plasmids, along with creating an approach that can use specifically place modified nucleotides for nucleic acid therapeutic based conjugation.

Results and Discussion Incorporation of biotinylated nucleotide and streptavidin binding to a plasmid DNA

Figure 1. Schematic showing modified nucleotide incorporated plasmid nanoparticle complex. A biotin-modified nucleotide is incorporated into a short DNA sequence using an 80-nt template in presence of DNA polymerase, then 1) that ds-sequence was ligated into a DNA plasmid as seen in step one. 2) In the second step, a nanoparticle conjugate with targeting ligands, stabilizing polymers (HASH-ADH), and a PEGylated streptavidin were mixed with the now biotinylated plasmid, creating a nanoparticle plasmid complex.



A two-part process was employed to strategically place the biotin-modified nucleotides into the plasmid where it did not potentially interfere with the specific regions essential for mRNA expression (Figure 1). First, the biotin-modified nucleotide (biotin-16-aminoallyl-2'-dCTP) was incorporated into an 80-nucleotide (nt) sequence that could be later ligated into a specific site of the EGFP plasmid. To incorporate the modified nucleotide, we replaced dCTP with a biotin-dCTP for primer extension using two different specifically designed templates. Both templates were flanked by the AseI restriction site with a 42-nt stretch in the middle for flexible positioning of the modified nucleotide. Template 1 had a single position for cytosine incorporation, while template 2 had two such positions. Figure 2A shows that the biotin-modified nucleotide can be incorporated and extended to the full-size product of the labeled primers for both templates 1 and 2. An oligonucleotide extension reaction with neither dCTP or biotin-modified dCTP resulted in shorter extended products, terminating at either 29 or 37-nt depending on the template, eliminating the possibility of mismatch incorporation when the fully extended product is formed in presence of either the biotin-modified dCTP or dCTP alone. As incorporation was deemed successful via primer extension, there was a question if the biotin-modified nucleotide may cause a reduction in endonuclease efficiency (Figure 2B). The control and biotin-modified nucleotide (BMN) incorporated products were incubated with AseI and denaturing polyacrylamide gel electrophoresis (PAGE) were used to detect the efficiency of enzymatic digestion (Figure 2B). Three major bands were detected in the digested lanes of both control and BMN products, which are the full uncut product (80-nt), a 1-cut product (69-nt) (where the labeled 5 is still available and the 3 side is cut) and the 5 cut end (11-nt) with the majority being of the later. It should be noted that there was no observable difference in the efficiency of the AseI digestion between the control and BMN sequences.


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Table 1. Templates for Biotin-dCTP incorporation via primer extension. The 80-nt templates (80-nt cut product length that is eventually inserted into the plasmid) had two distinct segments, i) AseI endonuclease restriction sites (yellow) and ii) 42-nt middle region for cytidine-biotin incorporation (red). The incorporation positions are in green, where templates were used with 1 and 2 possible incorporation positions.

A.

B.



Figure 2. Biotin-dCTP can be effectively incorporated into a short DNA sequence. A. Labeled primers were annealed to an 80-nt template and extended with nucleotide mixes with dCTP, biotindCTP, and no dCTP. The products were observed using denaturing PAGE. (Left) The template has 2 available positions for cytosine incorporation. (Right) The template has 1 available position for cytosine incorporation. B. Biotin incorporated sequences can be cleaved by endonuclease AseI. The purified 80-nt sequence with and without biotin-modified nucleotides was incubated with and without AseI. The reaction mixture was analyzed using denaturing PAGE. (Left) The sequence has 1 cytosine incorporation. (Right) The sequence has 2 cytosine incorporations. In all cases with the AseI addition, 3 bands were observed, the full 80-nt product, a 69-nt product, and an 11-nt product.

Upon successful design and synthesis of a BMN-EGFP plasmid, the conjugation point for nanoparticle complexation will be centered around streptavidin (nanoparticle anchor) and biotin (plasmid anchor) binding. To make sure this was feasible, and as a second examination of successful incorporation of the BMN via primer extension, control and BMN sequences of templates 1 and 2 (BMN-1 and BMN-2) were incubated with 2 to 1 molar equivalents of streptavidin to DNA. Binding was determined via a shift in the mobility of the band in PAGE


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(Figure 3A). The BMN template 2 was the only sample to show streptavidin-DNA binding through a slower migration of the band whereas all other samples showed no binding. This difference between BMN-1 and 2 templates was observed again as BMN sequences were incubated with increasing ratios of streptavidin to DNA. Figure S1 shows no apparent binding of streptavidin to BMN-1 as there is no migration difference observed when the no streptavidin lane and all other lanes with DNA and streptavidin were compared. On the contrary, the BMN-2 displayed a dosedependent binding where complete binding happened at a 2:1 ratio of streptavidin to DNA (Figure 3B). Two different populations of DNA-streptavidin conjugates were observed where a small population has 2 biotins bound to 1 streptavidin while the dominant population has 2 biotins to 2 streptavidin as seen by the slowest migrating band. As each monomer of the tetrameric quaternary structure of streptavidin has a binding pocket for biotin, we see that 2 BMNs results in successful binding of streptavidin to the full 80-nt BMN sequence. A.

B.



Figure 3. Biotin incorporated DNA sequence binds to streptavidin. A. A 10-molar excess of streptavidin was incubated with the 80-nt sequence with or without the biotin-modified nucleotides. In native PAGE a shift was observed with the 80-nt sequence that incorporated 2 biotin-modified nucleotides, which signified specific protein binding. B. Biotin incorporated sequence binds to streptavidin in a dose-dependent manner. Increasing concentrations of streptavidin were incubated with the 80-nt sequence that incorporated 2 biotin-modified nucleotides. In native PAGE a shift was observed with the 55-nt sequence starting at a 1:1 ratio.

Successful ligation of the biotinylated DNA segment into the EGFP plasmid Our goal was to devise a strategy to create a conjugation point at a precise location within the DNA plasmid, which instilled a strong bond not easily broken in cellular media or potentially in vivo. This was necessitated by the fact that previous attempts at creating plasmid nanoparticle complexes lacked specificity and stability. For example, PAMAM and other polymer plasmid nanoparticle conjugates have been previously reported, but these systems lacked any level of specificity in the number of conjugation points or more importantly the plasmid DNA is not effectively released from the polymer complex 23-25. By taking advantage of the many possible endonuclease sites (or the possibility of engineering sites into a plasmid sequence), a sequence can be ligated into a plasmid at a precise location, such that it would not interfere with functionally important DNA stretches. Also, as explained earlier, the sequence being ligated would have a defined number of conjugation points, thereby providing a precise control over the system. The AseI restriction site


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was chosen because it is almost 100 bp upstream of the CMV promoter in the EGFP plasmid that was being modified, with the expectation that it would not interfere with cellular machinery used to transcribe the plasmid DNA. This specific endonuclease was used due to the lack of cytosine within the 6-nucleotide digestion site, as it could not be predicted how the BMN would change the efficiency of endonuclease and ligase enzymatic activities. Figure S2 shows that after ligation of the BMN sequence into the EGFP plasmid, the ligated undigested plasmid migrates slower than the unmodified EGFP plasmid. Because the ligation happened at one specific site rather than between two cut sites, digested products of AseI could not be compared between the ligated and non-ligated products as the ligated sequence would just be recut out of the ligated plasmid. But this provided another important mechanism to validate successful ligation, as after purification the ligated plasmid was cut by AseI and compared with the ligated DNA sequence in an agarose gel (Figure 4). The gel clearly showed a small band migrating in line with a free 55-nt cut BMN sequence in the purified plasmid lane. These bands were enlarged and highlighted in the red box in Figure 4 where the left lane is the recut plasmid product. The newly digested plasmid also migrates similar to an AseI digested EGFP plasmid. All lanes with a cut plasmid were observed to move faster than the uncut plasmid which was uncommon, as the uncut plasmid is often supercoiled and thus migrates faster than the corresponding cut plasmid. The EGFP plasmid was cut with a number of other endonucleases with one cut site (SmfI, SmaI, PscI, and XbaI) (Figure S3). In spite of digesting with different enzymes the migration pattern that the digested plasmids migrate faster than the undigested remained unchanged. Finally, as ligation was established to be successful, the question still remained if plasmid was circularized or linear depending upon if both ends of the incorporated BMN sequence were ligated to the cut plasmid. The purified ligated plasmid product was subjected to restriction digestion by another endonuclease, XbaI, which is a



single cutter. We reasoned that if two sub 4.7 kb cut products were observed, then the plasmid would be considered as linear and only ligated at one of the BMN sequence ends instead of both ends. Figure S4 shows two bands, however, both bands run with control plasmid bands corresponding to circular and single cut plasmids. If the plasmid was non-circularized (linear), meaning only one end of the digested plasmid contained the introduced biotinylated sequence compared to both digested sites being re-ligated would result into ~1.5 kb and ~3.2 kb fragments. Because none of such fragments were observed, the newly formed BMN plasmid can be considered as circular meaning both ends of the 80 nt fragment were ligated to the parent EGFP plasmid. Also, the BMN-plasmid band migrated slightly slower, potentially providing additional proof of the successful ligation of the BMN sequence as the new plasmid is slightly larger and contained two biotin residues than the unligated EGFP plasmid.


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Figure 4. AseI endonuclease digestion resolved small ligated sequence in the BMN modified EGFP plasmid. Purified ligated plasmid and EGFP plasmid were cut with AseI and analyzed on a 1.5% agarose gel. Cutting of the ligated plasmid resulted in a band of the same size as the cut 55-nt. This band was contrasted and enlarged for better visualization. The data indicated that the 55-nt sequence with the biotin-modification was ligated into the plasmid and after purification could be detected and isolated.

Nanoparticle conjugation to biotinylated EGFP plasmid For assembly of a nanoparticle complex with the BMN-plasmid for potential targeted cellular uptake, an AuNP complex was synthesized that could be conjugated to the BMN-plasmid and induce targeted uptake into CD44 overexpressing tumorigenic cells. Gold nanoparticles were used as the central hub for the attachment of many modified polymers, which were prepared separately. This approach creates a situation where many polymers can be a part of the full complex, but only at two specific locations is conjugation occurring on the plasmid. The complex’s components that were conjugated to the gold surface included a PEGylated streptavidin, adipic dihyrdazide (ADH) modified hyaluronic acid (HA-ADH), and a PEGylated DNA based CD44 aptamer. The AuNP, HA-ADH, and PEGylated CD44 aptamer synthesis were described in a previously published report 26.

The streptavidin enabled the conjugation of the nanoparticle to the biotin modified EGFP

plasmid. Based on the in vitro streptavidin-biotin binding studies shown in figure 3, we could potentially bind two streptavidin per incorporated biotin modified nucleotide sequence. We intended each binding position to host a separate nanoparticle complex, so we added only 1.5 PEGylated streptavidin per nanoparticle. An excess of nanoparticles were incubated with the modified BMN-plasmid to try and facilitate two nanoparticles per each plasmid conjugation. Having two nanoparticles instead of one should provide the complex with more availability of



ligands to be incorporated for potential protection and targeting purposes. The BMN-plasmid could potentially be protected by the HA-ADH polymer which replaces the protected polymers used in other reports 22, 27. The ADH modification of HA also can reduce the number of negative charges on the naturally occurring HA, enabling potential interactions between the HA polymer and the plasmid. Finally, the CD44 aptamer was used to target cells overexpressing the CD44 receptor which is a common feature in many types of cancers, such as triple negative breast cancer (TNBC) 28,29,

hepatocellular carcinoma30, colon31, 32, head and neck33, and ovarian34, 35, along with cancer

stem cells 26, 36. Conjugated aptamers and antibodies had been shown to allow larger complexes to pass through the cell membrane via receptor-mediated endocytosis, giving the plasmid an avenue into the cell 37, 38. All polymers are thiolated for facile conjugation to the AuNP surface and also for the subsequent smart release mechanism. This has been previously shown, as HASH and PEGylated conjugates separate from the AuNP based on elevated glutathione levels inside the cell which can induce ligand exchange at the AuNP surface 26. This provides another advantage to this complex, as the majority of the components will separate from each other, outside of the biotin and streptavidin which has been already been mentioned to be strategically placed away from important transcription elements and thus its remaining on the site of conjugation will not hamper transcription.

Atomic Force Microscopy (AFM) was performed to generate an image of the entire complex. Figures 5A, S5 and S6 are AFM images of BMN-plasmid, the biotinylated plasmid-streptavidin nanoparticle complex (BPSNC), and the streptavidin nanoparticle conjugate respectively. The BMN-plasmids were observed to be 500 nm in length. This is the same length as the plasmidnanoparticle complexes, exhibiting the ability to not add to the size even after the nanoparticle was


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added. The selected plasmid shows what is believed to be three nanoparticles covering the plasmid sequence, a total height of ~90 nm. This is distinctly different than the nanoparticles on their own as the height of the individual nanoparticles are ~40 nm. We think the additive height of multiple nanoparticles sitting on the binding site of the plasmid gives a characteristic height increase seen in all BMN plasmid-nanoparticle complexes. This was observed again in Figure S7 where software was used to find the topological size and length of the nanoparticle conjugates and BMN plasmidnanoparticle complexes. The lone nanoparticles have heights ranging from 30-50 nm while the plasmid complexes are 70-90 nm. The addition to the plasmid DNA height of the nanoparticles could be one reason for the two height ranges, but we believe that the close proximity of the biotins within the BMN-plasmid leads to two nanoparticles sitting on top of each other giving this height characteristic. These three species were also subjected to a nanoanalyzer to measure the zeta potential and hydrodynamic diameter (Figures 5B and C, Table S1). The nanoparticles contained a small positive charge as the HASH-ADH is considered to be a zwitterionic polymer, while also containing a negatively charged aptamer and neutral conjugated protein. This zwitterionic HAADH polymer could also potentially play a role similar to PEI or other zwitterionic polymers for endosomal escape

39-42.

Zwitterionic polymers can induce a proton sponge effect as endosomes

acidify in the conversion to lysosomes, leading to osmotic swelling

43, 44.

The plasmid itself is

highly charged as expected. As the two are complexed together, only a minor decrease in the zeta potential is observed, suggesting that the addition of the nanoparticle complex causes charge interactions shielding a small population of the negatively charged phosphate backbone. The hydrodynamic diameters saw a similar trend to the AFM analysis. The nanoparticle complex was shown to be roughly 20 nm in diameter, while the plasmid was 125 nm and the BPSNC was approximately 100 nm in size. This again details the possible ability of the plasmid condensing



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around a zwitterionic polymer of the nanoparticle, slightly reducing the size. It should be noted that a large difference is observed between the AFM and DLS size results of the individual plasmid and the BPSNC. It is believed that the difference in experimental conditions could have led to this difference in sizes between AFM and DLS. This difference in plasmid sizing was seen in a number of other reports highlighting characterization of plasmid DNA using DLS and AFM, where the physical sizes of similar length plasmids matched our findings

45-50.

A salt solution in DLS can

lead to the shielding of the negative charges of the phosphate backboard, therefore reducing repulsive forces and potentially creating a smaller complex. Within the AFM procedures, the samples were prepared in water and dried on a silicon sample plate where with evaporation, the charge of the droplet can increase and the individual particles will have a high net negative charge, creating the possibility of the larger physical size of the plasmids 51. Although this difference in physical size based on experimentation was observed, the overall trends remained the same from DLS to AFM, establishing that the nanoparticle complex does not add substantial size to plasmid and the DLS values are ideal for the EPR effect in the case of tumor targeting 19, 52.

A.


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B.

C.

Figure 5. Characterization of nanoparticle and plasmid complex. A. Atomic force microscopy of ligated EGFP plasmid and streptavidin nanoparticle complex. B. Zeta potential and C. DLS of components of ligated EGFP plasmid-streptavidin nanoparticle complex. The full nanoparticle (with PEGylated streptavidin and aptamer conjugates), the EGFP plasmid and full plasmid nanoparticle complex were measured for net charge and hydrodynamic size. Samples were measured 5 times using a nanoanaylzer and results were compiled.



Nanoparticle-plasmid complexation is required for cellular uptake and expression of EGFP

As mentioned earlier, naked plasmid DNA is notorious for minimal cellular uptake because of the high negative net charge, very large molecular size, and susceptibility to enzymatic degradation. The nanocomplex is expected to shield the negative charge of the DNA while using a CD44 targeting aptamer for enhanced uptake into MDA-MB-231 TNBC cells. Initially, confocal fluorescence microscopy was used to assess delivery efficiency by visualization of the expressed EGFP protein. The CD44-BPSNC and Mutant-BPSNC treatments were examined first (Figure 6A). With CD44 versus the mutant aptamer being the only difference between the two complexes, a drastic change in EGFP associated fluorescence was observed, as the majority of cells treated with the CD44-BPSNC had greater signal while the mutant aptamer complex had no visible uptake, establishing the ability for the CD44 aptamer to facilitate cellular uptake most likely via receptor-mediated endocytosis resulting in the delivery of the EGFP plasmid. The EGFP mRNA was later quantified in cells treated with the CD44 and mutant CD44 complexes respectively, confirming the transcription of the EGFP plasmid in cells (Figure 6B), obviously without uptake transcription would not be feasible. The CD44-BPSNC treatment had an 84-fold increase in mRNA expression compared to the mutant-BPSNC.

Other nanoparticle-plasmid formulations were also assessed in cellulo to determine the efficiency and importance of nanoparticle plasmid conjugation via the biotin and streptavidin interaction. Cells with positive signals were counted along with the total number of cells within the frame to generate a measure of the delivery efficiency (Figure S9). When the transfected control in Figure S8 of the BMN-EGFP plasmid treatment is compared to the CD44-BPSNC, the


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nanoparticle complex had an efficiency of approximately 50%, while the artificial plasmid transfection (lipofectamine 2000) control had an efficiency of about 28% when plasmid amount was kept the same (150 ng). The low amount of plasmid used is likely to have contributed to the relative low lipofectamine transfection efficiency but highlights the ability of the CD44-BPSNC to deliver the plasmid in cells. Incidentally, no significant uptake was detected when visualizing results of treatments with other controls, such as EGFP plasmid no transfection, EGFP+CD44nano- streptavidin, EGFP+Mutantnano- streptavidin (Figure S8). The CD44 nanocomplex and EGFP co-treatment had very limited uptake (1%), showing that charge-charge interactions were not enough for viable cellular uptake. The EGFP plasmid on its own displayed no uptake, which is expected because of the aforementioned negative traits when not transfected or carried by other vehicles. There are a number of ongoing clinical trials for non-viral DNA vectors, making the drastic difference in EGFP expression efficiency detailed here an exciting step towards improved therapeutic options 35. The drastic difference in uptake between the CD44-BPSNC complex and other EGFP complexes details the importance of the biotinstreptavidin binding resulting in attachment of the plasmid on the nanoparticle for efficient cellular uptake.

In comparison to other work, the BPSNC is consistent with cited reports in producing GFP protein expression. Wong et al created a stimuli-responsive supramolecular complex for the targeted delivery GFP plasmid which released plasmid from the complex in lower pH environments such as the endosome and lysosome 53. In targeted cells, the complex displayed 35% GFP expression. Another study used a chitosan nanoparticle for the dual delivery of siRNA and plasmid DNA to ovarian cancer cells where the transfection efficiency of the plasmid was



20% 15. The exterior of the complex can also play large roles in GFP expression as a rougher, spiked filled PEI+silica nanoparticle exhibited improved expression (~50-80%) compared to softer and/or smoother exteriors (20-50%) 27. The PEI used in Song et al, along with a number of other reports, plays an important role in endosomal escape as the positive charge of the polymer induces a “proton sponge” effect. Both of these aspects, material roughness and charge, can be important details when choosing a polymer to further improve the efficiency of the BPSNC. In the aforementioned reports, treatments vary from 500-1000ng/ml to even high concentrations, but comparable or improved EGFP expression efficiency was found in this report at the low concentrations of 100 and 450 ng/ml treatments signifying the possibility for improved efficiency of non-viral DNA vector delivery using this methodology.

A.


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B.

Figure 6. Expression EGFP in TNBC cells shows targeted uptake of CD44 aptamer streptavidin nanoparticle-biotin modified EGFP plasmid complex. A. Cells were transfected with the EGFP plasmid or treated with either a mutant aptamer streptavidin nanoparticle-biotin modified EGFP plasmid complex or CD44 aptamer streptavidin nanoparticle-biotin modified EGFP plasmid complex. After 48 hours of treatment, cells were observed with a confocal fluorescence microscope at a 10x and 40x magnification. The CD44 targeted nanoparticle-biotin



modified EGFP plasmid treated cells had a considerable increase in fluorescence compared to the transfected EGFP plasmid treated cells. The mutant aptamer containing nanocomplex had no visible fluorescence indicating that most likely the uptake mechanism of receptor-mediated endocytosis is necessary for uptake of the large charged complex (Scale bars are 50 and 10 m respectively) . B. Expression of EGFP in TNBC cells increases 84-fold using targeted CD44 aptamer streptavidin nanoparticle-biotin modified EGFP plasmid complex. Cells were treated with either a mutant aptamer streptavidin nanoparticle-biotin modified EGFP plasmid complex or CD44 aptamer streptavidin nanoparticle-biotin modified EGFP plasmid complex. After 48 hours of treatment, EGFP mRNA was quantified to verify expression of the delivered plasmid but also compared the targeted and non-targeted complexes.

Conclusion Plasmid-based gene therapy can be an extremely effective therapeutic tool but the delivery of these large molecules is associated with many obstacles based on the limitations, such as size and high charge density. Nanoparticle systems have been created to overcome these limitations but inherently added to the overall, lacked in conjugation specificity, and have limited release of therapeutic payload, all reducing potential efficacy. We have created a flexible and minimalistic approach in improving upon plasmid-nanoparticle drug delivery design by incorporating biotinmodified nucleotides into a short sequence that we can ligate at a defined site into the plasmid. These nucleotides play host as a beacon for conjugation to a streptavidin conjugated nanoparticle. This design has two major advantages 1) the specificity of the conjugation position can be engineered to the exact nucleotide(s) within the plasmid, providing with incredible control in conjugation precision and 2) the nanoparticles have the ability to work as scaffolds to load individually or in combination any type of polymer (charge and size can be manipulated), drug, siRNA, targeting ligands, and diagnostic molecules among the many other possibilities. Within the complexes that were created, we were able to demonstrate that the addition of a PEGylated CD44 aptamer ensured targeted uptake of an EGFP plasmid compared to mutant aptamer complex


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as seen both in confocal microscopy and an 84-fold increase in mRNA expression. We believe that this plasmid-nanoparticle strategy, along with the use of incorporating modified nucleotides, could be useful for the targeted delivery of gene manipulating plasmids such as CRISPR/Cas9 and other protein-encoding genes opening the door to endless options for new treatments across a number of different diseases.

Materials and Methods High molecular weight hyaluronic acid was purchased from Glycosan Biosystems (Salt Lake, UT). Bifunctionalized

Polyethene

glycol

with

thiol

and

amine

functionalities,

N-(3-

(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), sodium borohydride, deuterium oxide (D2O), and Bovine Hyaluronidase were purchased from Sigma-Aldrich (USA). Adipic dihydrazide and Auric chloride (HAuCl3) were purchased from Fischer Scientific (USA). RPMI 1640 medium with L-glutamine, Dulbecco’s Modified Eagle’s Medium (DMEM) with 4.5 and 1 mg/ml L-glutamine, and fetal bovine serum (FBS) were purchased from Worldwide Medical Supplies (USA). CD44 directed DNA aptamer, the randomized version of the aptamer sequence, template sequences, and primers were purchased from Integrated DNA Technologies (IDT) (USA). Biotin-modified nucleotide (Biotin-16Aminoallyl-2'-dCTP) was purchased from TriLink Biotechnologies. DNA polymerase 1(Klenow) was bought from Thermo Scientific. The Milli-Q water used in all experiments was obtained from a three-stage Millipore Milli-Q plus 185 purification system (Millipore Corporation, USA) with a resistivity greater than 18.2 MΩ cm.

Thermocycler elongation of primers for biotin modified nucleotide incorporation



Single-stranded template sequences consisting of three regions were designed: 1) the first endonuclease cut site, 2) Biotin-dCTP incorporation sites, and 3) the second endonuclease cut site. Templates for EGFP plasmid delivery are listed in Table 1. Each cut site is flanked by 10 nucleotides. Primers were extended using the Klenow fragment, the large subunit of the DNA polymerase 1, incorporating a dNTPs mix that contained either dCTP or the biotin-modified dCTP using the primer 5-GCGAATGCAATTAATGCC-3. The newly formed double-stranded DNA was purified by denaturing PAGE. This process was repeated using 5-labeled primers. The 5′-end-radiolabeled single-stranded oligonucleotide primers were prepared and the radiolabeled DNA was visualized as reported previously in Beals et al 26.

Binding of biotin modified nucleotide incorporated sequence Control and biotin incorporated sequences labeled with γ-32P were either incubated with a 10-fold excess of streptavidin to biotin and binding were visualized using PAGE to observe a migration shift of the band representing the protein bound state. The same biotin modified sequences were incubated in increasing concentrations starting at a 1:50 streptavidin to visualize the binding efficiency of streptavidin to the biotin modified sequence. The samples were analyzed using PAGE.

Ligation of biotin DNA sequence into the EGFP plasmid Both the double-stranded DNA and the EGFP plasmid were cut by the endonuclease AseI, using the digestion condition recommended in the manufacturer’s protocol. Successful digestion was observed by PAGE using γ-32P labeled sequences. After heat deactivation of the enzymes, the resulting reactions were purified by a 3 kDa MWCO centrifuge tube and dried using a speed


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vacuum dryer. The separated solutions were reconstituted in 5 l of water, mixed and 10 l of 2x sticky end ligation buffer was added. The solution was mixed and gently shaken for an hour. The newly ligated plasmid was visualized and purified by band incision after analyzing on a 1.5% agarose gel. Ethidium bromide staining was used to image the gels in a UV Kodak imager system.

Detection of the 80-nt ligated to the plasmid The 80-nt ligated EGFP plasmid was purified, ligated and then was subjected to XbaI endonuclease digestion using manufacturer’s protocol and visualized on a 2 % agarose gel. A difference in migration was assessed for sequence incorporation. The EGFP purified ligated plasmid and 55-nt sequences were also subjected to AseI endonuclease cutting using manufacturer’s protocol and visualized on a 1.5% agarose gel. Ethidium bromide staining was used to image the gels on a UV Kodak imager.

Preparation of Gold Nanoparticles Gold nanoparticles were prepared accordingly to a previously published procedure 26. Particle size and stability were then determined by TEM and UV-vis spectrophotometry (Figures S11S12). A known molar extinction coefficient was used to obtain nanoparticle concentration via UV-vis spectrophotometry 26.

Preparation of Thiolated Low Molecular Weight Hyaluronic Acid The low molecular weight HA, which had an average size of 10 kDa, was prepared following a previously published protocol 26. The level of end thiolation of HA was then determined using Ellman’s method, a colorimetric assay used to detect free thiols, according to the manufacturer’s



protocol (Thermo Scientific).

Conjugation of Modified Thiolated Hyaluronic Acid to Adipic Dihydrazide End thiolated HA (HASH) was modified by reacting with adipic dihydrazide using a previous report 26. A set of analytical techniques, such as, NMR, Infrared Spectrometry and UV-Vis Spectrophotometry were performed to determine if the product matched as was reported previously.

DNA Aptamer Conjugation to Modified Polyethylene Glycol The 3-amine functionalized CD44 thioaptamer (CCA*A*GGCCTGCA*A*GGGA*A*CCA*A*GGA*CA*CA*G/3AmMO/) and mutant aptamer (NNNNNNNNNNNNNNNNNNNNNNNNNNNNN/3AmMO/) were purchased from IDT. The conjugation of the DNA aptamer and the PEG was performed according to a previously published report 26. 10 kDa MWCO centrifuge tubes were used to separate any unreacted PEG or DNA. Successful conjugation was established by using a gel shift assay.

Polyethylene Glycol Streptavidin Conjugation Streptavidin was mixed at a 1.5:1 molar ratio (PEG to streptavidin) with an excess of EDC and NHS in water over 24 hrs at room temperature to perform the conjugation reaction. The PEG conjugated aptamer was purified via 10 kDa MWCO centrifuge tubes.

Synthesis of the Nanocomplex 1 nmol of AuNP (100 l) and 1.5 nmol of PEG-aptamer (CD44 or mutant) and PEG-Streptavidin


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were added and shaken for 15 min. 100 l of a 5 mg/ml solution of HASH-ADH was added to the solution. The nanocomplex was allowed to shake overnight at 4C for complete conjugation. A 100,000 kDa MWCO centrifuge tube was used to separate unconjugated polymers from the nanocomplex.

Gold nanoparticle, hyaluronic acid, and PEGylated aptamer conjugation to plasmid The nanocomplex (100 pmols) was shaken with biotin-modified and unmodified ligated plasmids (500 ng) for 1 hour at room temperature.

Atomic Force Microscopy of nanoparticle-plasmid conjugate Images were acquired in the tapping mode under ambient conditions with a Multimode and NanoScope V controller (Digital Instruments, Bruker, US) using NSC15 silicon probes (Mikromasch, US). The probes have a resonance frequency between 250 and 400 kHz, nominal spring constant of 42 N m−1 and nominal tip radius of 10 nm. Amplitude set-points were established for 70 to 90% of the cantilever free amplitude. Topographic height images were obtained at a scan rate of 1 Hz.

Dynamic Light Scattering (DLS) and Zeta Potential Measurements The hydrodynamic diameters and zeta potentials were obtained according to the procedures reported in a previously publication 26. The samples were measured by using a Nanopartica Nanoparticle Analyzer SZ-100 (Horiba Scientific).

Cellular Uptake Detection by Confocal Fluorescence Microscopy MDA-MB-231 cells were seeded at a density of 10,000 and 25,000 cells per well respectively in



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an 8-well chamber slide and allowed to adhere overnight. The cells were subjected for 24 hrs to media containing 150 ng of either EGFP plasmid, transfected EGFP plasmid, CD44 aptamer EGFP plasmid, CD44 aptamer biotin-modified EGFP plasmid, mutant aptamer EGFP plasmid, and mutant aptamer biotin-modified EGFP plasmid. The cells were washed three times with full growth media and immediately analyzed for intracellular EGFP expression using a confocal microscope.

Quantitative RT-PCR MDA-MB-231 cells were seeded overnight at a density of 200,000 cells per well in 6-well plates. Cells were then treated for 48 hrs with media containing 150 ng of either CD44 and mutant aptamer nanoparticle biotin modified EGFP nanoparticle complexes. The cells were then washed three times with full growth media and total cellular RNA was obtained from treated cells using trizol reagent as per manufacturer’s protocol. The procedures for the synthesis of the cDNA and quantification of the qRT-PCR data were performed accordingly to a previously published report 54

. The primers for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and EGFP were

GAPDH:

sense-

5-AGCCACATCGCTCAGACAC-3

antisense-

5-

GCCCAATACGACCAAATCC-3 and EGFP: sense 5-AGGGCTATGTGCAGGAGAGA-3, antisense- 5-CTTGTGGCCGAGAATGTTTC -3). The relative mRNA levels were estimated by the comparative Ct method (Livak method) 55, 56.

ASSOCIATED CONTENT Supporting Information. The following files are available free of charge.


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brief description (file type, i.e., PDF) brief description (file type, i.e., PDF) S1, streptavidin binding to sequence with one biotin; S2, agarose gel of ligated plasmid; S3, restriction digestion of EGFP plasmid; S4, single digestion of EGFP and ligated EGFP plasmid; S5, AFM of BMN EGFP plasmid; S6, AFM of streptavidin conjugation nanoparticles; S7, AFM of BPSNC and size analysis; S8, Fluorescent images of plasmid-nanoparticle controls and lipofectamine transfected EGFP plasmid; S9, histogram of transfection efficiency of various plasmid – nanoparticle complexes and lipofectamine transfected EGFP; Table S1, DLS and Zeta potential of nanoparticle complexes

AUTHOR INFORMATION Corresponding Author *Email: [email protected] Author Contributions Experiments were performed by NB and NK. Data analysis was performed by NB and SB. Manuscript was written by NB and SB. ABBREVIATIONS HA, hyaluronic acid; DLS, dynamic light scattering; HASH, thiolated hyaluronic acid; HASH, thiolated hyaluronic acid; HASH-ADH, thiolated hyaluronic acid- adipic dihydrazide; ADH, adipic dihydrazide; TNBC, triple negative breast cancer; PEG, polyethene glycol; AFM, atomic



force microscopy; BPSNC, biotinylated plasmid-streptavidin nanoparticle complex; BMN, biotin modified nucleotide

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Graphic Abstract


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Efficient delivery of plasmid DNA using incorporated nucleotides for

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