materials Communication Study and Preparation of Multifunctional Poly(L-Lysine)@Hyaluronic Acid Nanopolyplexes for the Effective Delivery of Tumor Suppressive MiR-34a into Triple-Negative Breast Cancer Cells Jamila Djafari 1 , Javier Fernández-Lodeiro 1,2 , Hugo M. Santos 1,2 , Julia Lorenzo 3, * , Sergi Rodriguez-Calado 3 , Emilia Bértolo 4 , José Luis Capelo-Martínez 1,2 and Carlos Lodeiro 1,2, * 1 BIOSCOPE Group, LAQV@REQUIMTE, Chemistry Department, NOVA School of Science and Technology, NOVA University Lisbon, Caparica Campus, 2829-516 Caparica, Portugal; j.djafari@fct.unl.pt (J.D.); j.lodeiro@fct.unl.pt (J.F.-L.); hmsantos@fct.unl.pt (H.M.S.); jlcm@fct.unl.pt (J.L.C.-M.) 2 PROTEOMASS Scientific Society, Rua dos Inventores, Madam Parque, Caparica Campus, 2829-516 Caparica, Portugal 3 Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Campus Universitari, 08193 Bellaterra, Barcelona, Spain; sergi.rodriguez12@gmail.com 4 Section of Natural and Applied Sciences, Canterbury Christ Church University, Canterbury CT1 1QU, UK; emilia.bertolo@canterbury.ac.uk * Correspondence: julia.lorenzo@uab.es (J.L.); cle@fct.unl.pt (C.L.)  Received: 21 September 2020; Accepted: 18 November 2020; Published: 24 November 2020  Abstract: Non-viral gene delivery using exogenous microRNAs is a potential strategy for fighting cancers with poor prognosis and which lack specific therapies, such as triple-negative breast cancer (TNBC). Herein we report the synthesis of six nontoxic electrostatic polymeric nanocapsules (P1 to P6) for microRNA delivery in TNBC cells. 1H Nuclear Magnetic Resonance (NMR) spectroscopy and Scanning Electron Microscopy (SEM) were used to characterize the nanopolyplexes, synthesized with Poly(L-Lysine) and hyaluronic acid (Ha). Studies on the activity of the ternary HA/PLI/miRNA-34 nanopolyplexes towards TNBC cell line MDA-MB-231 were conducted. The nanopolyplexes mediated intracellular restoration of tumor suppressor miR34a was evaluated by using Western blotting to quantify the expression level of the Bcl-2 protein. The results suggest that the P5, with a ratio PLI/Ha of 0.05, was the most promising for the delivery of miR-34a into TNBC cells; the P5 nanocapsules were able to reduce Bcl-2 expression at a protein level, and had an effect in the overall cell viability after 24 h treatment. Keywords: polyplexes RNA; Poly-Lysine; triple negative breast cancer 1. Introduction Breast cancer is the second leading cause of cancer-related death after lung cancer, and is the most common diagnosed malignancy among women worldwide [1]. Significant progress has been made in diagnosing, monitoring and treating breast cancer. However, further research is needed to find ways to combat tumor relapses related to the onset of metastasis and resistance to conventional drugs; there is a need to develop new anticancer agents to promote the effectiveness of chemotherapy and reduce relapses [2,3]. Non-viral gene delivery using exogenous microRNAs (miRNAs) has emerged as a potential strategy for fighting against cancer. This kind of therapy has gained much attention, especially for Materials 2020, 13, 5309; doi:10.3390/ma13235309 www.mdpi.com/journal/materials Materials 2020, 13, 5309 2 of 9 more aggressive cancers which have worse prognosis and lack specific therapies, as is the case of the triple-negative breast cancer (TNBC). MiRNAs are conserved non-coding RNAs that negatively regulate gene expression by binding to the complementary sequence in the 30 -untranslated region (UTR) of target genes. MiR-34a has been involved in the inhibition of cell cycle progression and apoptosis of tumor cells; it also induces endothelial progenitor cell senescence and impedes angiogenesis [4]. In breast cancer, overexpression of miR-34a induces apoptosis of tumor cells, inhibits their proliferation and migration and reduces chemoresistance [5]. MiRNAs are short single-stranded sequences with unprotected 30 -hydroxy and 50 -phosphate ends, resulting in easy degradation by ribonucleases [6]; thus, they are only transiently expressed and have relatively short half-lives. Combined with a lack of tumor targeting, these are major limitations for their use in cancer therapy. Recently, nanocarriers have been suggested as a way to protect and target-deliver miRNAs at high levels directly to the tumor cells [7]. The ideal nanoparticle delivery vehicle requires the design of vectors sufficiently stable and nontoxic to transport the miRNA to the cytoplasm of the cancer cells. Many vectors have been developed based on the self-assembly of positively charged liposomes and polymers with the negatively charged nucleic acids, forming charged lipoplexes or polyplexes [8]. The small RNA miR-34a has been previously encapsulated in polyplexes formed by Poly-L-Lysine grafted with imidazole moiety and coated with PEGylated lipid vesicles [7]. The polyplexes, formed by self-assembly through electrostatic forces, yielded excellent results in terms of gene transport, non-degradation and intracellular delivery efficiency into gastric cancers cells [7]. The same research group also used this type of polyplexes to encapsulate Bcl-xL-specific shRNA-encoding plasmid DNA, targeting gastric cancer cells. Kim and co-workers designed and synthesized polyplexes formed by imidazole-poly-L-Lysine that were coated with hyaluronic acid to target gastric cancer cells through CD44, a glycoprotein over-expressed at their surface [2]. These electrically formed capsules can compact the nucleic acid and preserve it from the environment, increasing the delivery in the CD44- overexpressing tumor cells. The presence of imizadole groups allows the polyplexes to be sensitive to the pH of the medium. In fact, imidazole residues, which have a pka of 6, have a buffer capacity in the endosomal medium and can rapidly dissociate and release the nucleic acids in cancer cells. Herein we present the synthesis of six new nontoxic electrostatic polymeric capsules for miR-34a delivery. The miR-34a-containing nanopolyplexes, formed by imidazole-poly-L-Lysine and hyaluronic acid, were developed to target cancer cells through the overexpressed CD44 receptor. The study of the impact of the nanopolyplexes for miR-34a delivery was also studied in a cellular model of triple-negative breast cancer. 2. Materials and Methods Poly(L-Lysine hydrobromide) (PLL, Mw 10,000 Da) was purchased from Alamanda Polymers. N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), 1-hydroxybenzotriazole hydrate (HOBt), 4-Imidazoleacetic acid, Sodium Hydroxide (NaOH), Hyaluronic acid sodium salt from Streptococcus equi (1.5–1.8 × 106 Da), 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) were purchased from Sigma Aldrich without further purification. Dialysis tubing benzoylated (cut off 2000) was purchased from Sigma Aldrich. Water was ultra-pure grade (type I). MISSION® microRNA Mimic (has-miR-34a-5p) was purchased from sigma Aldrich. The miR-34a sequence was: UGGCAGUGUCUUAGCUGGUUGU. 2.1. Synthesis of Imidazoled Poly-L-Lysine (PLI) Poly-L-Lsine graft imidazole (PLI) was synthesized using N-(3-dimethylaminopropyl)-N-ethyl carbodiimide hydrochloride (EDC)/1-hydroxy benzotriazole hydrate (HOBt) coupling method from Poly(L-Lysine hydrobromide) (PLL), by modification of a published method [2,8]. The molar ratio of carboxylic acid to ε-amine used was 0.50. 4-Imidazoleacetic acid (0.29 mmol) was dissolved in 10 mL of MilliQ water. Then, two molar excess of HOBt and EDC were added to the solution. The pH of the Materials 2020, 13, 5309 3 of 9 reaction was adjusted to pH 5 with 1N NaOH and let under stirring for 30 min. An aqueous solution of PLL (0.58 mmol of ε-amine, Mw 10,000, DPn 48) was added to the reaction, and the pH adjusted to pH 8.5. The reaction was left under stirring for 24 h at room temperature. Subsequently, the reaction was dialyzed against repeated MilliQ water changes using a dialysis membrane with a molecular weight cutoff of 2000, in order to remove the excess of reagents. The resulting solution was lyophilized and stored at −20 ◦ C until use. 1 H NMR PLI (400 MHz, D O, ppm): δ = 7.80 − 7.29 (m, CH imidazole group), 4.18 (s, α–CH), 2.81 2 (m, ε–CH2 ), 1.82–1.05 (s, β–CH2 , γ–CH2 and δ–CH2 in Poly-L-Lysine). 2.2. Preparation and Characterization of Ternary Polyplexes HA/PLI/MiRNA-34 Ternary polyplexes were prepared by electrostatic interaction modifying the N/P ratio in each experiment, following published methods [2,3,9]. N/P ratio corresponds to the moles of amine groups in each polymer (N) per moles of phosphate groups in miRNA-34a; N/P ration has an essential influence in the transfection efficiency [10]. PLI/miRNA-34a polyplexes were prepared in 10 mM HEPES buffer (pH 7.5). A fixed amount of miRNA-34a (3 µg) was mixed with PLI stock solution (1 mg/mL) at N/P ratios of 5, 10 and 20, and incubated at room temperature for 20 min. Then, a solution of hyaluronic acid (2 mg/mL) in 10 mM HEPES buffer (pH 7.5) was mixed with PLI/miRNA-3a polyplexes at 0.05 and 0.1 molar ratios of HA to PLI and incubated at room temperature for 20 min. 2.3. Scanning Electron Microscopy (SEM, Quanta650) The SEM Quanta650 was used to examine the surface morphology of the synthesized polyplexes. For sample preparation, the polyplexes were diluted 1:10 in MilliQ H2 O and mounted on a metal stub with an aluminum adhesive tape. After drying overnight, a sputtering device was used to coat the nanoparticles with a 5 nm width platinum layer under high vacuum. Samples were then analyzed with a 10 kV electron beam. Gel retardation assays were conducted to evaluate the condensation ability of ternary HA/PLI/miRNA-34a polyplexes. The polyplexes contained 0.5 µg of miRNA-34a, and were prepared at N/P ratios of 5, 10 and 20. Electrophoresis was carried out with 1% agarose gel (w/v) containing ethidium bromide (0.05 µg/mL) in Tris-acetate-EDTA (TAE) buffer at 100 V for 30 min. The retardation of polyplexes was visualized under a UV lamp. 2.4. Cell Cytotoxicity Assay Human triple-negative breast cancer cells MDA-MB-231 were cultured in RPMI media supplemented with 10% FBS (Gibco-BRL), and incubated at 37 ◦ C in a humidified atmosphere with 5% CO2 . Cells were seeded into a 96-well plate at a cell density of 3.0 × 103 cells per well and, then incubated for 24 h before the addition of the nanopolyplexes at 100 nM of encapsulated miRNA-34a. The growth inhibitory effect was measured after 24 h treatment employing the PrestoBlue assay [11]. PrestoBlue (10 µL; resazurin-based solution) was added to each well. After a two-hour incubation period (37 ◦ C, 5 % CO2 , 98 % humidity), the fluorescence was quantified using a fluorescent multilabel plate reader (Victor3, PerkinElmer) with excitation at λ = 531 nm and recorded at λ = 572 nm. Cell cytotoxicity was evaluated in terms of cell growth inhibition in treated cultures and expressed as a percentage of the control conditions. Each experiment was repeated at least three times, and each concentration was tested in at least three replicates. 2.5. Evaluation of the In Vitro Bcl-2 Suppression by MiR-34a Delivery The nanopolyplexes mediated intracellular restoration of tumor suppressor miRNA-34a was evaluated by quantifying the expression level of Bcl-2 protein by Western blotting. MDA-MB-231 cells were seeded (2 × 105 cells per well) in 6-well plates and cultured for 24 h to reach 70% confluence at the time of transfection. Next day, the different polyplexes were added to each well at a final miRNA-34a concentration of 100 nM. After 72 h incubation, the medium was removed, cells were rinsed twice with Materials 2020, 13, 5309 4 of 9 Materials 2020, 13, x 4 of 10 1harvested and lysed with M-PER mL of phosphate-buffered lysis 10 saline (PBS, buffer mM,(Thermo Scientific, pH 7.4), and finally,Waltham, cells were MA., USA)and harvested following the lysed with manufacturer’s indications. After centrifugation at 12,000× g for 10 min, the supernatants M-PER lysis buffer (Thermo Scientific, Waltham, MA, USA) following the manufacturer’s indications. were collected, After and the at centrifugation concentrations of min, 12,000× g for 10 proteins were measured the supernatants wereusing Bradford’s collected, and the reagent (Bio-Rad concentrations of Laboratories, USA). The protein samples were denatured by boiling for 5 min and proteins were measured using Bradford’s reagent (Bio-Rad Laboratories, USA). The protein samples loaded onto SDS- PAGE were gel for electrophoresis. denatured by boiling for 5The minproteins and loadedwere transferred onto SDS-PAGE onto gelPVDF (Polyvinilidene for electrophoresis. difluoride) The proteins membranes (Millipore, Darmstadt, Germany), and then incubated in blocking solution were transferred onto PVDF (Polyvinilidene difluoride) membranes (Millipore, Darmstadt, Germany), (5% non-fat dried milk) at room temperature for one hour. The anti-Bcl-2 (Cell Signalling Technology, and then incubated in blocking solution (5% non-fat dried milk) at room temperature for one hour. Danvers, MA,anti-Bcl-2 The USA) was(Celladded into blocking Signalling solutionDanvers, Technology, and incubated MA, USA)at 4 °C overnight. was added intoTheblocking membranes were solution subsequently and incubatedincubated with the The at 4 ◦ C overnight. secondary goat anti-mouse membranes antibodyincubated were subsequently conjugated with with theHRP for 1 h. secondary Protein expression was normalized against β-actin expression. Blotting images goat anti-mouse antibody conjugated with HRP for 1 h. Protein expression was normalized againstwere acquired with the VersaDoc β-actin imaging expression. systemimages Blotting (Bio-Rad, Hercules, were CAwith acquired USA) and the analyzedimaging VersaDoc by the software provided system (Bio-Rad, by the manufacturer. Hercules, CA, USA) and analyzed by the software provided by the manufacturer. 3. Results 3. Results and and Discussion Discussion Figure 11 depicts Figure depictsthethe keykeystepssteps involved in the in involved formation of miRNA-34a the formation containing polyplexes. of miRNA-34a containing The Imidazole polyplexes. poly-L-lysine The Imidazole (PLI) was(PLI) Poly-L-Lysine synthesized by conjugation was synthesized of carboxylic by conjugation acidacid of carboxylic of of 4- Imidazoleacetic acid with ε-amine of poly-L-lysine at a molar ratio of 0.5. We obtained 4-Imidazoleacetic acid with ε-amine of Poly-L-Lysine at a molar ratio of 0.5. We obtained a co-polymer a co-polymer composed of composed of two two different different repetitive repetitive units, units, the the lysine lysine and and the the imidazole imidazole moiety, moiety, attached attached through through amidelinkages. amide linkages. H1 1 H NMR NMR analysis analysis confirmed confirmed the the successful successful conjugation conjugation ofof the the imidazole imidazolegroup groupin inthe the polymer by the presence of imidazole protons peaks at 7.80–7.29 ppm, similar to polymer by the presence of imidazole protons peaks at 7.80–7.29 ppm, similar to the literature. the literature. Figure 1. Schematic diagram of the key steps involved in the formation of the stable miRNA-34a/PLI/HA Figure 1. Schematic diagram of the key steps involved in the formation of the stable miRNA- ternary polyplexes, showing the protonability at the endosomal environment, which allows intracellular 34a/PLI/HA ternary polyplexes, showing the protonability at the endosomal environment, which gene delivery. allows intracellular gene delivery. The percentage of imidazole moiety linked was calculated by NMR using characteristics imidazole The percentage of imidazole moiety linked was calculated by NMR using characteristics proton α–CH peaks integration [12] (See Figure 2). We obtained a percentage of 46.9% of imidazole imidazole proton α–CH peaks integration [12] (See Figure 2). We obtained a percentage of 46.9% of moiety for an input of 0.5%. The polymerization degree of the initial Poly-L-Lysine (PLI) is about imidazole moiety for an input of 0.5%. The polymerization degree of the initial Poly-L-Lysine (PLI) 48 units with an average weight of 10,000 Da (g/mol). The copolymer obtained has an average is about 48 units with an average weight of 10,000 Da (g/mol). The copolymer obtained has an average composition of 22 imidazole units and 26 free lysines units, with an approximate molar mass of 8.5 kDa. composition of 22 imidazole units and 26 free lysines units, with an approximate molar mass of 8.5 MiRNA-34a (3 µg) was encapsulated in PLI at different N/P ratio and covered with HA (see results, kDa. Section 2.2). Polyplexes were formed by electrostatic forces between the negative charges of RNA, positive charges of PLI and negative charge of HA. At endosomal pH (pH < 6), the nitrogen atoms of imidazole residues were protonated, and protons accumulate in the polyplexes leading to the release of miRNA-34a. All the polyplexes synthesized are summarised in Table 1. Materials 2020, 13, 5309 5 of 9 Materials 2020, 13, x 5 of 10 1 H NMR Figure 2.Figure 2. 1H NMR spectrum spectrum of imidazolePoly-L-Lysine of imidazole poly-L-Lysine (PLI) in D (PLI) in2ODat room temperature (400 MHz, 2 O at room temperature (400 MHz, D2O, ppm). D2 O, ppm). MiRNA-34a (3 µ g) was encapsulated in PLI at different N/P ratio and covered with HA (see results, Section 2.2). Polyplexes Table 1. MiRNA-34a/PLI/HA were formed ternary by electrostatic polyplexes preparedforces withbetween variousthe N/Pnegative charges of and PLI/HA ratios, RNA, positive charges of PLI and negative charge of HA. At endosomal pH (pH < 6), the nitrogen in 10mM HEPES buffer. Each polyplex contained 3 µg of miRNA-34a. atoms of imidazole residues were protonated, and protons accumulate in the polyplexes leading to the release of miRNA-34a. Polyplexes All the polyplexes N/Psynthesized Ratio are summarised PLI/HA Ratio in Table 1. Table 1. MiRNA-34a/PLI/HAP1 5 prepared with0.05 ternary polyplexes various N/P and PLI/HA ratios, in P2 10mM HEPES buffer. Each polyplex contained53 µ g of miRNA-34a. 0.1 P3 10 0.05 Polyplexes N/P ratio PLI/HA Ratio P4 10 0.1 P5 P1 205 0.05 0.05 P6 P2 205 0.1 0.1 P3 10 0.05 P4 10 0.1 P5 The surface morphology of the polyplexes 20 examined was 0.05by scanning electron microscopy (SEM); P6 20 0.1 results are shown in Figure 3. We can observe the spherical morphology of the nanoplexes is maintained in all the synthetic conditions. The surface morphology However, the different of the polyplexes ratios used was examined affect the by scanning size microscopy electron of the polyplexes. P5, with (SEM); a higher N/P ratio (20) shows a substantial size increase compared with results are shown in Figure 3. We can observe the spherical morphology of the nanoplexes P1–P4 (seeis Table 2). Unfortunately, the high maintained in all N/P and PLI/HA the synthetic ratiosHowever, conditions. of P6 made the sample the different not ratios suitable used forsize affect the SEM of preparation. the polyplexes. The changes P5,added in size with a higher to theN/P ratio (20) in variations shows the acomposition substantial sizeofincrease compared with the polyplexes couldP1–P4 affect their (see Table 2). Unfortunately, the high N/P and PLI/HA ratios of P6 made the sample not suitable for biological activity. P5 polyplex capsules, with an N/P ratio of 20 and PLI/HA ratio of 0.05, have an SEM preparation. The changes in size added to the variations in the composition of the polyplexes average diameter could affectof 468.6 their nm. activity. P5 polyplex capsules, with an N/P ratio of 20 and PLI/HA ratio biological of 0.05, have an average diameter of 468.6 nm. Table 2. Polyplexes synthesised, average size (nm) and errors. Standard Error of the Mean Polyplexes Average Diameter (nm) (SEM) P1 121.46 6.02 P2 121.30 13.49 P3 125.41 5.30 P4 118.90 9.12 P5 448.07 36.68 P1 121.46 (SEM) 6.02 P1 P2 121.46 121.30 6.02 13.49 P2 P3 121.30 125.41 13.49 5.30 P3 P4 125.41 118.90 5.30 9.12 Materials 2020, 13, 5309 P4 P5 118.90 448.07 9.12 36.68 6 of 9 P5 448.07 36.68 Figure 3. SEM images of the polyplexes at the different formulations. Average diameter and Figure3.3.SEM SEM polydispersity Figure images index images of theofwas (PDI) thecalculated polyplexes polyplexes at SEM at thefrom the formulations. different different images offormulations. theAverage samples. Average diameter diameter and and polydispersity polydispersity index (PDI) was index (PDI)from calculated wasSEM calculated imagesfrom SEM of the images of the samples. samples. The N/P ratio of the polyplexes influences their condensation ability and transfection efficiency. The TheTheN/PN/Pratio condensationratioof of the thepolyplexes ability polyplexes influences of the ternaryinfluences their their condensation condensation PLI/HA/miRNA-34a ability ability andwas polyplexes transfection evaluated efficiency. efficiency. by gel The The condensation condensation ability of ability the of ternary the PLI/HA/miRNA-34a ternary polyplexes PLI/HA/miRNA-34a was evaluated polyplexes retardation assay (see Figure 4). For P5 and P6, which have the highest quantities of PLI and HAwas by gel evaluatedretardation by gel of assay (see retardation all Figure 4). For assay studies the polyplexes P5 (see Figureand (N/P4).P6, which For=P5 ratio have and 20), the highest P6, whichwas miRNA-34a quantities haveretarded,of PLI the highest and HA of quantities indicating all the thatofthe PLI polyplexes and HA of miRNA-34a studies all the was (N/P ratio polyplexes completely = 20), miRNA-34a studies condensed (N/P in PLA =was ratioand 20), HA.retarded, miRNA-34a indicating that the was retarded, For polyplexes P1–P4, miRNA-34a indicating with was thatof N/P ratio the completely miRNA-34a 5 (P1 and P2) condensed was in completelyPLA and HA. condensed For in polyplexes PLA and HA.P1–P4, For with N/P polyplexes ratio of P1–P4, 5 (P1 withand and 10 (P3 and P4), the quantities of PLI and HA were not enough to condense fully miRNA-34a N/PP2) and ratio of105 (P3 (P1 and andP4), P2) in the andquantities 10 (P3 and polyplexes. of P4), PLI and HA were not the quantities enough of PLI to condense and HA were notfully miRNA-34a enough in the to condense polyplexes. fully miRNA-34a in the polyplexes. Gel retardation Figure 4. Gel retardation assay assay of of the ternary polyplexes P1–P6, which possess different N/P N/P ratios. ratios. Figure 4. Gel retardation assay of the ternary polyplexes P1–P6, which possess different N/P ratios. To investigate whether the difference in the N/P ratios and the HA concentration can effectively change the efficacy of miRNA-34a in silencing Bcl-2 expression, we first evaluated the cytotoxic effect of the different formulation of the nanopolyplexes in the triple negative breast cancer cell line MDA-MB-231; these cells were chosen because they overexpress the CD44 receptor. As shown in Figure 5, the lower N/P ratios (5 and 10) showed no inhibition of in vitro cell growth compared to the control group. The cells treated with a N/P ratio of 20 had little effect in cell inhibition growth, with cell viabilities around 80%. These results suggest that an increasing concentration of imidazole can protect the miRNA-34a delivery by buffering the endosomal medium and facilitating its delivery. of the different formulation of the nanopolyplexes in the triple negative breast cancer cell line MDA- MB-231; these cells were chosen because they overexpress the CD44 receptor. As shown in Figure 5, the lower N/P ratios (5 and 10) showed no inhibition of in vitro cell growth compared to the control group. The cells treated with a N/P ratio of 20 had little effect in cell inhibition growth, with cell viabilities around 80%. These results suggest that an increasing concentration of imidazole can Materials 2020, 13, 5309 7 of 9 protect the miRNA-34a delivery by buffering the endosomal medium and facilitating its delivery. N/P molar ratio 5 10 20 HA/PLI molar ratio 0.05 0.1 0.05 0.1 0.05 0.1 125 100 Cell viability (%) 75 50 25 0 P1 P2 P3 P4 P5 P6 Polyplex Figure 5. Effect Figure on cell 5. Effect viability on cell ofofmiRNA-34a-loaded viability nanopolyplexes miRNA-34a-loaded nanopolyplexes onon triple triple negative negative breast breast cancer cancer MDA-MB-231 MDA-MB-231 cells. Cells were treated for 24 h with the indicated polyplex; N/P and HA/PLI molarmolar cells. Cells were treated for 24 h with the indicated polyplex; N/P and HA/PLI ratiosratios for each polyplex for each areare polyplex included includedininthe thegraph. Thecell graph. The cellsurvival survival was was determined determined byPrestoBlue by the the PrestoBlue method as a percentage of the control. Data are expressed as the mean ± standard error of method as a percentage of the control. Data are expressed as the mean ± standard error of experimentsexperiments performed in triplicate. performed in triplicate. Bcl-2Bcl-2 has been has been identified identified asas a potentregulator a potent regulator ofof apoptosis apoptosisbyby preventing preventing thethe permeability permeability of theof the mitochondrial outer membrane [13]. An overexpression of Bcl-2 confers mitochondrial outer membrane [13]. An overexpression of Bcl-2 confers cancer cells’ resistance cancer cells’ resistance to to chemotherapeutics agents such as doxorubicin, making it an exciting target chemotherapeutics agents such as doxorubicin, making it an exciting target for further cell sensibilization for further cell sensibilization to current treatments [Error! Bookmark not defined.]. Bcl-2 is targeted by miRNA- to current treatments [12]. Bcl-2 is targeted by miRNA-34a, and there is an epigenetic inactivation 34a, and there is an epigenetic inactivation of miRNA-34a in cells derived from breast cancer [14,15]. of miRNA-34a in cells derived from breast cancer [14,15]. In this work, the expression of Bcl-2 In this work, the expression of Bcl-2 was evaluated at the protein level after the delivery of miRNA- was evaluated at the protein 34a in triple-negative level breast canceraftercells thebydelivery of miRNA-34a the nanopolyplexes. in triple-negative Untreated cells were used breastas cancer a cells negative by the nanopolyplexes. control. Results are Untreated shown in Figurecells6.were Modestusedandasnon-downregulation a negative control. Results of Bcl-2 are shown expression in Figure 6. Modest was observed andcells in the non-downregulation treated with P2 and P4. of Bcl-2 P2 andexpression P4 have high was observed PLI/HA in the ratio (0.1) butcells low treated to with medium P2 and P4. P2 and molecular N/PP4ratios have(5high and 10PLI/HA ratio (0.1) respectively). but low The results fortoP2medium molecular and P4 suggest N/P ratios that higher (5 andpresence of HA in the 10 respectively). coating The mayfor results confer P2 andan excessive P4 suggest negative electrostatic that higher charge presence oftoHA thein complex, the coating resulting may confer aninexcessive lower transfection negativeefficiency of thecharge electrostatic nanopolyplexes and decreasing to the complex, resultingmiRNA-34a uptake. in lower transfection Remarkable efficiency downregulation ofand of the nanopolyplexes Bcl-2 expressionmiRNA-34a decreasing was noted inuptake. the cells Remarkable treated with P1 and P3, both of downregulation 0.05 PLI/HA ratio and 5 and 10 N/P molar ratios, respectively. The best result for downregulating Bcl-2 expression was noted in the cells treated with P1 and P3, both 0.05 PLI/HA ratio and 5 and 10 N/P Bcl-2 are from P5 and P6, which have the highest N/P ratio (20) and can completely condense the molar ratios, respectively. The best result for downregulating Bcl-2 are from P5 and P6, which have miRNA, suggesting that they are the best nanopolyplexes to deliver the miRNA-34a into the cells the highest (FigureN/P 6b). ratio (20) and can completely condense the miRNA, suggesting that they are the best nanopolyplexes to deliver The results suggestthe thatmiRNA-34a into the the HA helps in targeting cells (Figure cells via CD446b).recognition, but an equilibrium The results suggest that HA helps in targeting the cells via CD44 between the two polymers is needed for a correct internalization of the nanopolyplexes. recognition, but an equilibrium Furthermore, between the two of the presence polymers a higher is needed molar ratiofor a correct in of imidazole internalization the nanoparticles of the cannanopolyplexes. Furthermore, overcome the deficiency of RNA delivery the presence in the cells of a higher dueratio molar to theof complete imidazole condensation of the miRNA. in the nanoparticles P6overcome can and especially the P5 are deficiency of RNApromising delivery nanopolyplexes in the cells due for to thethe delivery completeof miRNA-34a condensation into triple-negative of the miRNA.breast P6 and cancer cells; P5 especially are promising nanopolyplexes for the delivery of miRNA-34a into triple-negative breast cancer cells; both nanopolyplexes were capable of reducing Bcl-2 expression at a protein level and had an effect in overall cell viability after 24 h treatment. This is a very promising result that could make cancer cells more sensitive to chemotherapy or radiotherapy; this is of special relevance for TNBC patients, who tend to have a higher recurrence rate after diagnosis, a short disease-free interval, and reduced overall survival, especially due to the lack of targeted therapies [16]. The use of mi-RNA 34a in TNBC models has previously been reported as showing a reduction in the angiogenic process and in the proliferation, migration and onset of apoptosis or senescence. For effective miRNA replacement therapy, miRNAs must avoid early clearance and degradation in the bloodstream, travel through the extracellular matrix of the tumor, enter the target tumor cell and induce the silencing to enable gene regulation [17,18]. In this work, we loaded miR34a Materials 2020, 13, 5309 8 of 9 Materials 2020, 13, x 8 of 10 into PLI/HA nanopolyplexes to overcome these delivery challenges. Future studies should determine both nanopolyplexes the potential therapeuticwere capable efficacy of reducing Bcl-2 expression of nanopolyplexes P1, P6 andat specially a protein level and P5, in had an effectwith combination in overall cell viability after 24 h treatment. traditional antitumoral drugs, using in vivo models. Figure 6. Protein Figure expression 6. Protein levels expression of Bcl-2 levels in the of Bcl-2 MDA-MB-231 in the MDA-MB-231 cells byby cells Western Westernblot analysis. blot analysis.Cells Cellswere treated fortreated were 24 h with for 24the nanopolyplexes. h with the nanopolyplexes. (a) (a) The Bcl-2 The Bcl-2protein levelwas protein level wasdetermined determined by Western by Western blotting using blotting anti-Bcl-2 using antibody; anti-Bcl-2 antibody;N/PN/P and HA/PLImolar and HA/PLI molar ratios ratios for each for each polyplex polyplex are included are included in the in figure.(b) the figure. (b) Graph Graph showing showing the therelative Bcl-2 relative abundance Bcl-2 calculated abundance as a percentage calculated of the control. as a percentage of theEach control. Each value valuerepresents representsthe mean the mean± S.E.M. fromfrom ± S.E.M. duplicate determinations. duplicate determinations. 4. Conclusions This is a very promising result that could make cancer cells more sensitive to chemotherapy or radiotherapy; this is of special relevance for TNBC patients, who tend to have a higher recurrence We have successfully synthesized six new poly(L-lysine) polyplexes P1 to P6, containing rate after diagnosis, 1a short disease-free interval, and reduced overall survival, especially due to the encapsulated miR-34a. lack of targeted H NMR therapies anduse [16]. The SEM analyses34a of mi-RNA confirm in TNBCthe formation models of the polyplexes has previously been reportedand the ratio of as L-Lysine showing apresent. reductionCytotoxic studies process in the angiogenic of the six andpolyplexes, using with in the proliferation, humanand migration triple-negative onset of breastapoptosis cancer cell or senescence. For effective miRNA replacement therapy, miRNAs must avoidP1 line MDA-MB-231 yielded promising results, especially for polyplex and P5, early to a lesser extent, clearance and P6. P1 and P5 degradation arebloodstream, in the promising nanopolyplexes travel through thefor specific delivery extracellular matrix ofof themiRNA-34a tumor, enter the target tumor into triple-negative breast cell and cells. cancer induceThe the polyplex silencing to enable was ablegene regulation to reduce Bcl-2[17]. In this work, expression at a we protein level,loaded showing miR34a into PLI/HA an effect nanopolyplexes in the overall to overcome cell viability after 24 these delivery These h treatment. challenges. Future results studies suggest there is should determine the potential therapeutic efficacy of nanopolyplexes P1, P6 and specially potential to use this non-toxic vector for selective miR-34a delivery in CD44 overexpressing tumors. P5, in combination with traditional antitumoral drugs, using in vivo models. Further studies with capsules P1, P5 and P6 are in progress. 4. Conclusions Author Contributions: Conceptualization, C.L., J.D. and J.L.; methodology, J.D., J.F.-L., H.M.S., J.L. and S.R.-C.; validation, We J.D.,have J.F.-L.,successfully H.M.S., J.L. and S.R.-C.; formal synthesized six new analysis, J.D., J.F.-L., poly(L-lysine) H.M.S., S.R.-C. polyplexes P1 toandP6,J.L.; investigation, containing J.D., J.F.-L., H.M.S., S.R.C. and 1J.L.; resources, C.L., J.L.C.-M., J.L.; writing—original draft preparation, J.D., C.L. and encapsulated miR-34a. H NMR and SEM analyses confirm the formation of the polyplexes and the J.L.; writing—review and editing, J.D., J.F.-L., H.M.S., J.L., E.B. and S.R.-C.; Final draft editing, E.B.; Supervision, ratio of C.L., J.F.-L., L-Lysine J.L.C.-M. and present. Cytotoxic J.L.; project studies of J.D., administration, the six polyplexes, C.L., usingacquisition, J.F.-L.; funding with human triple-negative C.L., J.L.C.-M., H.M.S., J.L. Allbreast authorscancer havecell read line MDA-MB-231 and agreed to theyielded publishedpromising versionresults, especially for polyplex P1 and P5, to of the manuscript. a lesser extent, P6. P1 and P5 are promising nanopolyplexes for specific delivery of miRNA-34a into Funding: We thank the financial support by the PROTEOMASS Scientific Society (Portugal) (General Funding Grantstriple-negative 2018–2019, asbreast well as cancer cells. The polyplex the Associate Laboratory wasResearch able to reduce Unit forBcl-2 expression Green at a protein level, Chemistry—Clean Processes showing an effect in the overall cell and Technologies—LAQV-REQUIMTE viability financed byafter 24 h treatment. national funds fromThese results(UID/QUI/50006/2013) FCT/MEC suggest there is and co-financed potential to by usethethisERDF non-toxicunder thefor vector PT2020 Partnership selective Agreement miR-34a delivery (POCI-01-0145-FEDER—007265). in CD44 overexpressing tumors. We also thankstudies Further for thewithfinancial capsulessupport P1, P5FCT—Fundação para a Ciência e Tecnologia within the R&D Units and P6 are in progress. Project Scope: UIDB/04077/2020. J.D.; C.L.; J.F.-L. and J.L.C.-M. thanks to the FCT-MEC the research grant (PTDC/QEQ-MED/2118/2014). J.F.-L. thank FCT/MEC (Portugal) the junior researcher contract under DL57 Author Contributions: Conceptualization, C.L.; J.D. and J.L.; methodology, J.D.; J.F.-L.; H.M.S.; J.L. and S.R.-C. programme. All authors thanks to the FCT, for the Project PTDC/QEQ-MED/2118/2014 for financial support, and J.D. validation, J.D.; J.F.-L.; H.M.S.; J.L.; and S.R.-C. formal analysis, J.D.; J.F.-L.; H.M.S., S.R.-C., and J.L.; for her researcher contract. H.M.S is funded by the FCT Investigator Program 2015 (IF/00007/2015). J.L. and S.R.C. thanksinvestigation, to the ProjectJ.D.; MICINNJ.F.-L.; (Spanish H.M.S., S.R.C., Ministryandof J.L.; resources, Science, C.L.; J.L.C.-M.; Innovation J.L. writing—original and Universities) draft RTI2018-098027-B-C22. preparation, J.D.; C.L. and J.L.; writing—review and editing, J.D.; J.F.-L.; H.M.S.; J.L., E.B. and S.R.-C. Final draft Conflicts of Interest: editing, The authors E.B. Supervision, C.L.;declare J.F.-L.; no conflictand J.L.C.-M. of interest. J.L.; project administration, J.D.; C.L. J.F.-L.; funding acquisition, C.L.; J.L.C.-M.; H.M.S.; J.L. All authors have read and agreed to the published version of the manuscript. References 1. Kamangar, F.; Dores, G.M.; Anderson, W.F. Patterns of cancer incidence, mortality, and prevalence across five continents: Defining priorities to reduce cancer disparities in different geographic regions of the world. J. Clin. Oncol. 2006, 24, 2137–2150. [CrossRef] [PubMed] Materials 2020, 13, 5309 9 of 9 2. Kim, E.; Yang, J.; Kim, H.-O.; An, Y.; Lim, E.-K.; Lee, G.; Kwon, T.; Cheong, J.-H.; Suh, J.-S.; Huh, Y.-M.; et al. Hyaluronic acid receptor-targetable imidazolized nanovectors for induction of gastric cancer cell death by RNA interference. Biomaterials 2013, 34, 4327–4338. [CrossRef] [PubMed] 3. Deng, X.; Cao, M.; Zhang, J.; Hu, K.; Yin, Z.; Zhou, Z.; Xiao, X.; Yang, Y.; Sheng, W.; Wu, Y.; et al. Hyaluronic acid-chitosan nanoparticles for co-delivery of MiR-34a and doxorubicin in therapy against triple negative breast cancer. Biomaterials 2014, 35, 4333–4344. [CrossRef] [PubMed] 4. Zhao, T.; Li, J.; Chen, A.F. MicroRNA-34a induces endothelial progenitor cell senescence and impedes its angiogenesis via suppressing silent information regulator 1. Am. J. Physiol. Endocrinol. Metab. 2010, 299, E110–E116. [CrossRef] [PubMed] 5. Misso, G.; Di Martino, M.T.; De Rosa, G.; Farooqi, A.A.; Lombardi, A.; Campani, V.; Zarone, M.R.; Gullà, A.; Tagliaferri, P.; Tassone, P.; et al. Mir-34: A new weapon against cancer? Mol. Nucleic Acids 2014, 3, e195. [CrossRef] [PubMed] 6. Bail, S.; Swerdel, M.; Liu, H.; Jiao, X.; Goff, L.A.; Hart, R.P.; Kiledjian, M. Differential regulation of microrna stability. RNA 2010, 16, 1032–1039. [CrossRef] [PubMed] 7. Kumari, P.; Ghosh, B.; Biswas, S. Nanocarriers for cancer-targeted drug delivery. J. Drug Targ. 2015, 24, 179–191. [CrossRef] [PubMed] 8. Tros de Ilarduya, C.; Sun, Y.; Düzgüneş, N. Gene delivery by lipoplexes and polyplexes. Eur. J. Pharm. Sci. 2010, 40, 159–170. [CrossRef] [PubMed] 9. Chan, L.C.; Cox, B.G. Kinetics of amide formation through carbodiimide/N—Hydroxybenzotriazole (HOBt) couplings. J. Org. Chem. 2007, 72, 8863–8869. [CrossRef] [PubMed] 10. Jang, E.; Kim, E.; Son, H.Y.; Lim, E.K.; Lee, H.; Choi, Y.; Park, K.; Han, S.; Suh, J.S.; Huh, Y.M.; et al. Nanovesicle-mediated systemic delivery of microRNA-34a for CD44 overexpressing gastric cancer stem cell therapy. Biomaterials 2016, 105, 12–24. [CrossRef] [PubMed] 11. Zhao, Q.-Q.; Chen, J.-L.; Lv, T.-F.; He, C.-X.; Tang, G.-P.; Liang, W.-Q.; Tabata, Y.; Gao, J.-Q. N/P ratio significantly influences the transfection efficiency and cytotoxicity of a polyethylenimine/chitosan/DNA complex. Biol. Pharm. Bull. 2009, 32, 706–710. [CrossRef] [PubMed] 12. Xu, M.; McCanna, D.J.; Sivak, J.G. Use of the viability reagent PrestoBlue in comparison with alamarBlue and MTT to assess the viability of human corneal epithelial cells. J. Pharm. Toxicol. Methods 2015, 71, 1–7. [CrossRef] [PubMed] 13. Lang, C.; Kiefer, C.; Lejeune, E.; Goldmann, A.S.; Breher, F.; Roesky, P.W.; Barner-Kowollik, C. Palladium-containing polymers via a combination of RAFT and triazole chemistry. Polym. Chem. 2012, 3, 2413. [CrossRef] 14. Youle, R.J.; Strasser, A. The BCL-2 protein family: Opposing activities that mediate cell death. Nat. Rev. Mol. Cell Biol. 2008, 9, 47–59. [CrossRef] [PubMed] 15. Inao, T.; Iida, Y.; Moritani, T.; Okimoto, T.; Tanino, R.; Kotani, H.; Harada, M. Bcl-2 inhibition sensitizes triple-negative human breast cancer cells to doxorubicin. Oncotarget 2018, 9, 25545–25556. [CrossRef] [PubMed] 16. Lodygin, D.; Tarasov, V.; Epanchintsev, A.; Epanchintsev, A.; Berking, C.; Knyazeva, T.; Körner, H.; Knyazev, P.; Diebold, J.; Hermeking, H. Inactivation of miR-34a by aberrant CpG methylation in multiple types of cancer. Cell Cycle 2008, 7, 2591–2600. [CrossRef] [PubMed] 17. Collina, F.; Di Bonito, M.; Bergolis, V.L.; De Laurentiis, M.; Vitagliano, C.; Cerrone, M.; Nuzzo, F.; Cantile, M.; Botti, G. Prognostic value of cancer stem cells markers in triple-negative breast cancer. Biomed Res. Int. 2015, 2015, 158682. [CrossRef] [PubMed] 18. Valcourt, D.M.; Day, E.S. Dual regulation of miR-34a and notch signalling in triple-negative breast cancer by antibody/miRNA nanocarriers. Mol. Ther. Nucleic Acids 2020, 21, 290–298. [CrossRef] [PubMed] Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).