Engineering of inhalable nano-in-microparticles for co-delivery of small molecules and miRNAs

Vol.:(0123456789) Discover Nano (2023) 18:38 | https://doi.org/10.1186/s11671-023-03781-0 1 3 Discover Nano Research Engineering ofinhalable nano‑in‑microparticles forco‑delivery ofsmall molecules andmiRNAs MarjanMotiei1· OndrejMišík2· ThanhHuongTruong1· FrantisekLizal2· PetrHumpolíček1· VladimírSedlařík1· PetrSáha1 Received: 7 October 2022 / Accepted: 27 January 2023 © The Author(s) 2023 OPEN Abstract In this study, novel Trojan particles were engineered for direct delivery of doxorubicin (DOX) and miR-34a as model drugs to the lungs to raise local drug concentration, decrease pulmonary clearance, increase lung drug deposition, reduce systemic side effects, and overcome multi-drug resistance. For this purpose, targeted polyelectrolyte nanoparticles (tPENs) developed with layer-by-layer polymers (i.e., chitosan, dextran sulfate, and mannose-g-polyethyleneimine) were spray dried into a multiple-excipient (i.e., chitosan, leucine, and mannitol). The resulting nanoparticles were first characterized in terms of size, morphology,in vitroDOX release, cellular internalization, andin vitrocytotoxicity. tPENs showed comparable cellular uptake levels to PENs in A549 cells and no significant cytotoxicity on their metabolic activity. Coloaded DOX/miR-34a showed a greater cytotoxicity effect than DOX-loaded tPENs and free drugs, which was confirmed by Actin staining. Thereafter, nano-in-microparticles were studied through size, morphology, aerosolization efficiency, residual moisture content, andin vitroDOX release. It was demonstrated that tPENs were successfully incorporated into microspheres with adequate emitted dose and fine particle fraction but low mass median aerodynamic diameter for deposition into the deep lung. The dry powder formulations also demonstrated a sustained DOX release at both pH values of 6.8 and 7.4. Keywords Nano-in-microparticles· Pulmonary delivery· Chitosan· Small molecules· miRNAs Abbreviations dA,theory Aerodynamic diameter APSD Aerodynamic particle size distribution APS Aerodynamic Particle Sizer ACI Andersen Cascade Impactor CS Chitosan DS Dextran sulfate DMSO Dimethyl sulfoxide DOX Doxorubicin DMEM Dulbecco’s Modified Eagle Medium * Marjan Motiei, [email protected] | 1Centre ofPolymer Systems, University Institute, TBU, Tr. Tomase Bati, 5678Zlin, CzechRepublic. 2Faculty ofMechanical Engineering, Brno University ofTechnology, Technicka 2896/2, 61669Brno, CzechRepublic. Vol:.(1234567890) Research Discover Nano (2023) 18:38 | https://doi.org/10.1186/s11671-023-03781-0 1 3 ρeff Effective density ED Emitted dose EE Encapsulation efficiency ECACC European Collection of Authenticated Cell Cultures FBS Fetal bovine serum FPF Fine particle fraction FITC Fluorescein isothiocyanate isomer I dG Geometric diameter GSD Geometric standard deviation NSCLC Human non-small cell lung cancer Leu Leucine LC Loading capacity Mant Mannitol Mans Mannose MMAD Mass median aerodynamic diameter M2-TAMs M2-like tumor-associated macrophages NIH/3T3 Mouse embryonic fibroblast cell line MDR Multi-drug resistance NPs Nanoparticles NSCLC Non-small cell lung cancer A549 Non-small human adenocarcinoma epithelial cell line OPA O-phthaldialdehyde PDMS Polydimethylsiloxane PEI Polyethyleneimine RPMI Roswell Park Memorial Institute SSPD Small-Scale Powder Disperser ρtap Tap density tPENs Targeted polyelectrolyte nanoparticles TB Tracheobronchial TPP Tripolyphosphate USP/Ph. Eur. United States Pharmacopeia/European Pharmacopeia Introduction The second most common carcinoma, lung cancer, is often treated with chemotherapy. However, effective cancerchemotherapy requires overcoming a number of drawbacks, including poor aqueous solubility, non-targeting ability, nonspecific distribution, systemic side effects, a limited therapeutic index, and multi-drug resistance (MDR) [1, 2]. A practical approach to address these issues is simultaneous delivery of chemotherapeutics and non-chemotherapeutics, which possesses additional benefits like lowering drug dosages and achieving synergistic therapeutic efficacy [3, 4]. However, few studies utilized nanosystems for co-delivery of small molecules [5–7] and small molecules/tumor suppressor genes [8, 9]; herein, for the first time, co-deliveryof model drugs such as doxorubicin (DOX) and miR-34a has been performed through targeted polyelectrolyte nanoparticles (tPENs) embedded in microparticles to combine the main beneficial characteristics of co-delivery of drugs, cell targeting ability, and low cytotoxicity. Doxorubicin (DOX), as a leading anticancer drug, interacts with DNA and causes cancer cell apoptosis. This amphiphilic drug is effective against several cancer types, such as lung cancer [10]. miRNAs are a class of highly conserved singlestranded non-coding RNAs with fast biodegradation and a short half-life rate. They can inhibit the post-transcriptional gene expression by binding to 3′ untranslated regions of mRNAs [11]. miR-34a has become one of the most effective miRNAs as tumor suppressors, and its expression is downregulated in some human cancers, like lung cancer [12, 13]. However, such a strategy needs an efficient intracellular delivery system due to the fast biodegradation and short halflife of miRNAs [14, 15]. Therefore, the design of inhaled carriers with deep lung deposition and efficient intracellular co-delivery of DOX and miR-34a is the major challenge that needs to be overcome. Vol.:(0123456789) Discover Nano (2023) 18:38 | https://doi.org/10.1186/s11671-023-03781-0 Research 1 3 Pulmonary delivery is a promising method for lungcancer therapy because of the large alveolar surface area, thin epithelial barrier, extensive vascularization, absence of hepatic first-pass effect, and low enzymatic activity of the lungs [10]. The respirable dry formulations should have aerodynamic diameters from 1 to 5µm to efficiently reach the deep lungs, swell upon deposition in the moist lung, and offer a sustained release through the matrix [16]. Particles larger than 5µm would deposit in the upper airways primarily due to the inertial impaction [17]. Particles between 0.1 and 1µm would be exhaled quickly, and particles smaller than 100nm would be highly deposited in all airways, mainly in upper areas by the diffusional mechanism [18]. Strategically, nano-in-microparticles (NIMs) can improve the therapeutic effects by releasing nanoparticles (NPs) into the deep lungs and avoiding macrophage uptake. NPs can also anticipate as valuable platforms for targeted controlled release and degradation resistance of drugs [19]. Due to high biodegradability and biocompatibility [20], versatility in encapsulating hydrophilic and hydrophobic drugs, and capacity to cross biological barriers [21], polymeric NPs have attracted the most attention among the various types of NPs in inhaled therapeutic systems. Herein, a new class of therapeutic NPs was synthesized as tPENs composed of chitosan (CS) core and Mannose (Mans)-g-Polyethyleneimine (PEI) shell cross-linked electrostatically through negatively charged dextran sulfate (DS). CS, a biocompatible, biodegradable, and antibacterial biopolymer, and PEI form stable nanocomplexes with polyanions such as tripolyphosphate (TTP) and DS through the protonation of amino groups below their pKa [22]. Due to the high expression of Mans receptors in human lung adenocarcinoma [23], and targeting both non-small cell lung cancer (NSCLC) cells and M2-like tumor-associated macrophages (M2-TAMs), PEI was functionalized with Mans [24]. Polyethyleneimine (PEI) also induces endosomal rupture by its “proton sponge effect” and facilitates the polyplexes’ release into cytosols [25]. After that, tPENs were micronized into a mucoadhesive excipient with low toxicity and readily degradable properties to increase the aerosolization efficiency and NPs’ uptake through the mucus layer [21]. CS, mannitol (Mant., a non-hygroscopic polymorphic acyclic sugar alcohol) [26], and leucine (Leu., hydrophobicamino acid) were the promising components of the excipient. Moreover, this study has focused on the engineering and the feasibility of innovative biocompatible NIMs based on CS for the synergetic delivery of DOX and miR-34a in lung cancer therapy using a spray-drying technology. The innovative NIMs were developed to combine the benefits of smart multifunctional NPs and the respirable microspheres. The resulting micronized powders were fully characterized in terms of their morphology, production yield, flowability, aqueous reconstitution, and aerosolization efficiency, which confer a controlled and sustained release of cargos. Particle uptake into non-small-cell lung cancer was studied in A549 cells and compared to NIH-3T3 cell line at 2h. Finally, the effect of DOXand miR-34a-loaded tPENs was also evaluated on cytotoxicity. Result anddiscussion FT‑IR analysis A simple nucleophilic addition reaction between the primary amine of PEI and the aldehyde group of Mans was used to create Mans-PEI, and two distinct methods confirmed this. As shown in Fig.1a, UV–Vis absorption spectra exhibited the modification of PEI with Mans by the presence of a new absorption peak at 340nm as opposed to PEI and Mans [27]. To further explore the linkage formation between PEI and Mans, FT-IR was utilized. As shown in Fig.1b, the characteristic absorptions of PEI at 2950, 2848, and 1471 cm−1 corresponded to the stretching and bending vibration of CH2 bonds, and the absorption peaks at 3297 and 1598 cm−1 belonged to the N–H bond [28]. Compared to the spectrum of PEI and Mans, the C=O stretching of Mans at 1654 cm−1 disappeared, and two strong absorption bands at 1457 and 1577 cm−1 were observed in the Mans-PEI spectrum assigned to the ring CC, and C-N stretches, respectively. In addition, the stretching vibration of C-O is located at 1052 cm−1, which reveals the presence of C–OH. Figure1c shows the FT-IR spectra of CS, TPP, DS, Mans-PEI, and tPENs. Two peaks in the FT-IR spectrum of CS powder were assigned at 898 and 1151 cm−1, which correspond to the structure of saccharides. The other peaks were assigned at 1066 cm−1 for the > CO-CH3 stretching vibration, 1378 cm−1 for the CH3 symmetrical deformation mode, 1658 cm−1 for the amide, 2900 cm−1 for the C-H stretching vibration, and 3378 cm−1 for the N–H symmetric stretching vibration. The bands at around 804 cm−1 obtained from the asymmetric S–O-S vibration and the asymmetric and symmetric SOO− stretching vibrations at 1230 cm−1 and 987 cm−1 confirmed the presence of the sulfate group in the DS spectrum [29]. The tPENs’ spectra analysis revealed peaks at 1117 cm−1 and 1147 cm−1, which were associated with P=O in TPP [22] and the production of sulfones [30], respectively. Additionally, tPENs displayed sharp bands with high intensities at Vol:.(1234567890) Research Discover Nano (2023) 18:38 | https://doi.org/10.1186/s11671-023-03781-0 1 3 924 cm−1, 1014 cm−1 (sulfo-group), 1410 cm−1 (symmetric stretching of COO −), 1570 cm−1 (Amide II band), 1637 cm−1 (C=O (amide I band)), 3286 cm−1 and 3178 cm−1 (O–H (H-bonded)), and 3421 cm−1 (primary amines). Therefore, it can be supposed that the polyplex has been achieved successfully. In accordance with Fig.1d, NH3+ vibrations are related to the spectra of Leu at 3079, 1608, and 1511 cm−1, and the consecutive bands at 1581 and 1407 cm−1 are attributed to the bending vibrations of the carboxyl group [31, 32]. The FT-IR spectrum of Mant shows strong stretching vibration peaks ofOH (3286 and 3388), CO (1020 and 1078), and CH (2908 and 2971). Additionally, at 1417 and 1282 cm−1, the OH in-plane bending and CH bending vibrations can be seen [33]. When comparing the NIMs spectra to that of the other bulks, it can be demonstrated that several bands at 3079, 1608, and 1511 cm−1 have vanished, while peaks at 1407 and 1573 cm−1 associated with carboxyl vibrations have intensified, and peaks at 923, 1014, 2900, and 3378 cm−1 have appeared. Therefore, it can be supposed that CS, Leu, and Mant are preponderantly adsorbed into the NIMs. tPENs characterization SEM and DLS were used to describe the morphology, size, size distribution, and surface charge of the tPENs. Figure2a indicates a SEM micrograph of uniform dispersed spherical tPENs with the size of 222.23 ± 54.88nm. DLS analysis demonstrated that the hydrodynamic size of the tPENs was around 241.99 ± 0.51nm with a relatively narrow distribution of 0.29 ± 0.01. The compact structure of the tPENs is attributed to pH, because the ionizable groups have a significant impact on the swelling behavior, network structure, and permeability of tPENs in response to pH fluctuation [34]. The presence of a sufficient positive charge on the tPENs’ surface (31.53 ± 4.21) also supports the colloidal stability of the nanostructures [35]. Fig. 1 UV–Vis absorption spectra of PEI, Mans, and Mans-PEI (a), FT-IR spectra of PEI, Mans, and Mans-PEI (b) tPENs composed of CS, TPP, DS and Mans-PEI (c), and NIMs containing tPENs, CS, Leu, and Mant Vol.:(0123456789) Discover Nano (2023) 18:38 | https://doi.org/10.1186/s11671-023-03781-0 Research 1 3 NIMs characterization Even though NPs have been suggested for effective drug delivery to the lung epithelium, the small size severely limits their application in pulmonary delivery because of high diffusional deposition (particle size ˂ 100nm), exhalation (100nm˂ particle size˂ 1µm), or lung phagocytic clearance [36, 37]. Herein, different formulations of NIMs were synthesized in various ratios of excipients (i.e., CS, Leu, and Mant) to efficiently deliver therapeutic agents to the respiratory system and address these issues. F1, F2, and F3 were first synthesized in tPENs/CSratiosof 1/1, 1/1.5, and 1/3, respectively. According to Fig.2b–d, the SEM micrographs showed that spherical particles with rough surfaces tend to be aggregated with increasing CS concentration. These deformations are probably caused by the spray-drying process and high repulsive forces among highly positive charged tPENs and CS. Therefore, F1 with a lower CS concentration and dG of 5.90 ± 0.57µm was selected for further experiments. After that, Leu was added to the excipient (i.e., F4, F5, and F6) to lessen NIM aggregation, delay the disintegration of the “interstitial bridges,” and decrease particle wetting [38, 39]. As shown in Fig.2e–g, significant interparticle coalescences were demonstrated at higher Leu concentrations, and the physical properties of F6 were difficult to describe (not reported in Table2). Some other studies also confirmed that crumpled particles are typical morphology of NIMs containing Leu [38]. Table2 also demonstrates that the presence of Leu led to a significantly higher amount of Carr’s index and a lower amount of yield. Among the formulations F4-F6, F4 by Carr’s index of 21.25 ± 5.30 and yield of 40.13 ± 1.91 was selected for thesubsequent development step. In the next step, Mant was added to the F4 formulation to improve the viscoelasticity, liquid content, and aqueous re-dispersibility [40]. New formulations (F7 and F8) demonstrated that Mant significantly affected dG, dA,theory, and yield, which agrees with Kho & Hadinoto, who confirmed that Mant was the most promising candidate of NIMs morphology but Fig. 2 Size distributions and SEM images of tPENs (a) and different formulations of NIMs including F1 (b), F2 (c), F3 (d), F4 (e), F5 (f), F6 (g), F7 (h), and F8 (i) Vol:.(1234567890) Research Discover Nano (2023) 18:38 | https://doi.org/10.1186/s11671-023-03781-0 1 3 not the aqueous re-dispersibility [37]. According to Fig.2h, i, lower Mant concentration led to the formation of spherical particles with smaller size distribution. Finally, F7 was selected as an optimized formulation for further experiments due to significantly higher yields, re-dispersibility, and smaller dG and dA,theory (Table1). Due to the critical impact of particles’ aerodynamic behavior on lung deposition, the aerodynamic behavior of the two formulations (F4 and F7) was analyzed using APS and ACI. The average MMAD of F7, the breathable fraction of an aerosol, was in the range of 5.88 ± 0.57 and 6.42 ± 0.35µm, and F4 was 5.87 ± 1.03 and 6.54 ± 0.05µm by APS and ACI, respectively (Table2). Most scientists strongly believe that particles larger than 5µm should not be administered to the lungs [41]. However, invivo measurements by Usmani etal. revealed that about 20% of 6µm particles inhaled at a higher flow rate of 60 LPM were deposited in central and intermediate airways [42]. This is also in good agreement with one-dimensional computational models of dry powder lung delivery with idealized geometry replicas created by Finlay [43] and Soong Table 1 Physical and aerosolization characteristics of inhaled dry powder n = 3, Mean ± Standard Deviation. The different letters in the same column indicate significant differences between the means (p value < 0.05), and the values marked with the same letters are not statistically different Fρ eff (g/mL) Carr’s index Sf/Si dG (µm) dA, theory Yield (%) F1 0.30 ± 0.02 15.22 ± 6.25a1.63 ± 0.24 5.90 ± 0.57a2.87 ± 0.68a48.46 ± 8.54a F4 0.32 ± 0.10 21.25 ± 5.30b1.49 ± 0.06 5.26 ± 0.73a2.67 ± 0.79a40.13 ± 1.91b F5 0.33 ± 0.05 20.68 ± 12.00b1.54 ± 0.11 5.12 ± 1.22a2.64 ± 0.84a40.11 ± 11.04b F7 0.33 ± 0.01 21.74 ± 7.59b1.46 ± 0.09 2.09 ± 0.58b1.07 ± 0.21b49.65 ± 4.58a F8 0.33 ± 0.03 23.08 ± 14.58b1.97 ± 0.68 2.40 ± 0.98b1.22 ± 0.34b25.20 ± 4.25c Table 2 Aerodynamic behavior evaluated by APS and ACI n = 3, Mean ± Standard Deviation. The different letters in the same column indicate significant differences between the means (p value < 0.05), and the values marked with the same letters are not statistically different F MMAD (µm) GSD (µm) FPF<5µm of APSD (%) FPF<5µm of ED (%) ED (%) F4 APS 5.87 ± 1.03 1.74 ± 0.06 38.79 ± 13.98a- - ACI 6.54 ± 0.05 2.16 ± 0.04 30.30 ± 0.59 0.26 ± 0.00 90.50 ± 3.54 F7 APS 5.88 ± 0.57 1.58 ± 0.02 37.15 ± 8.53b- - ACI 6.42 ± 0.35 1.77 ± 0.15 33.11 ± 4.46 0,71 ± 0.00 96.17 ± 4.68 Fig. 3 ACI deposition patterns of two spray-dried formulations (i.e., F4 and F7). Eight stages were analyzed fitting the particles cutoff diameter: stage 0 (˃8.6µm), stage 1 (6.5–8.6µm), stage 2 (4.4–6.5µm), stage 3 (3.2–4.4µm), stage 4 (1.9–3.2µm), stage 5 (1.2–1.9µm), stage 6 (0.55–1.2µm), stage 7 (0.26–0.55µm), and stage 8 (˂0.26) Vol.:(0123456789) Discover Nano (2023) 18:38 | https://doi.org/10.1186/s11671-023-03781-0 Research 1 3 etal. [44] at the inspiration flow rate of 70 LPM. These models predict that around 15% of 10µm particles are deposited in tracheobronchial (TB), and the optimal TB drug delivery is attained by particles with MMADof 2to5µm when 25% to 55% of such particles are deposited in the TB area. Moreover, the powder formulations prepared in this work can be a suitable drug carrier for treating the TB region, especially in the first generations of branching. According to Table2, the GSD of F7 and F4 is narrow, and 90.50 ± 3.54% of the applied dose from F4 and 96.17 ± 4.68% of F7 were dispersed into the impactor. FPFAPS and FPFACI also confirmed that more than 30% of the measured APSD were smaller than 5µm. The actual deagglomeration causes the difference in FPFAPS and FPFACI within the Breezhaler device. The non-ideal deagglomeration shifts APSD to larger particles and significantly reduces the FPF. Therefore, these results confirmed that the final efficacy depends on the specific inhaler device and its deagglomeration ability and formulation properties. In the ACI measurements, the Breezhaler (Novartis) was used for aerosol dispersion, a device with quite a simple deagglomeration technology, low resistance, and ease to use [45]. Figure3 displays the deposition patterns of the NIMs on an eight-stage ACI. A significant fraction of the aerosolized particles was collected in the mouthpiece adapter, the induction port, and the pre-separator, which signified the low FPF. However, the fraction of the aerosolized particles that ended up in stages 2, 3, and 4 were increased in F7 after adding Mant, confirming more effective agglomerate dispersion with slightly higher FPF than F4. Residual moisture content To verify the effectiveness of the dry powder, TGA assessed the residual moisture content of the particles and excipients [46]. Physically and chemically preadsorbed CO2, moisture, and other gases are the main causes of the frequent weight loss below 100°C. Therefore, thermogravimetric analysis of the samples was performed at 25–150°C. At temperatures between 25 and 150°C, Leu and Mant lost approximately 0.21% and 0.97% of their respective masses, and the moisture loss of tPENs was around 28.9% at a temperature of 61.66°C. Nonetheless, the major moisture loss of NIMs (2.61%) occurred at 134.76°C, which can be attributed to the hydrophobic Leu [47]. The chitosan’s hygroscopicity nature may lead to this high residual moisture [48], which results in additional size increases, lowering flowability, and reducing the long-term stability of DPI formulations [46]. It also affects the disposition of the drug particles at the point of deposition [49], and the ability to overcome the barriers related to lung geometry and the physiological conditions in the lungs [46]. miR‑34a andDOX loading efficiency andDOX invitro release study As demonstrated in Fig.4a, in contrast to the other bands, which display a bright well with no trailing band for the pellets and no bright band for the supernatant, naked mir-34a, the negative control, exhibits a sharp band. Therefore, PENs interacted successfully with mir-34a and inhibit migration of mir-34a into the gel, in agreement with data already reported [50]. In quantification of DOX loading efficiency, tPENs demonstrated EE of 64.38 ± 0.61% and a lowering amount of LC Fig. 4 Gel retardation assay of mir-34a in tPENs at N/P ratio of 150 (a), DOX release profiles from tPENs and NIMs at 37°C and two different pH values of 7.4 and 6.8 for 72h. Each bar represents the average of three experiments, and error bars refer to standard deviation (b) Vol:.(1234567890) Research Discover Nano (2023) 18:38 | https://doi.org/10.1186/s11671-023-03781-0 1 3 (6.9 ± 0.24%), which can be explained by the strong dependency of LC on the weight ratio of tPENs in accordance with Eq.(6). The cumulative release profile of both nanoand microparticles at two different pHs, physiological pH (7.4) and a more acidic pH (6.8) to mimic the lung-lining fluid of a lung cancer situation, is depicted in Fig.4b. The results showed a similar release profile for the tPENs at pH 6.8 (64.38 ± 1.80%) and pH 7.4 (66.2 ± 0.54%) after 72h, which can be discussed by high colloidal stability of tPENs [28]. For microformulation of F7, the DOX tends to release more sustainably than tPENs at both pHs, which is more pronounced at pH 7.4 (44.23 ± 2.27) than pH 6.8 (49.89 ± 2.13). It agrees with Lebhardt etal., who declared that larger particles demonstrate more sustained release in contrast to smaller ones because drug release relies on diffusion distance [40]. The higher drug release of NIMs at pH 6.8 can be explained by the pKa of CS (6.5) and its higher dissolution at pH 6.8 than 7.4. The prolonged-release profile, for at least 72h, points out that the carriers improve the likelihood that the drug encounters the target for a longer time and releases it into the desired place. In vitro cytotoxicity oftPENs Firstly, cytotoxicity studies were performed using tPENs, free drugs (i.e., DOX and miR-34a), and co-drugs-loaded tPENs against the A549 cell line by MTT assay. Human non-small cell lung cancer (NSCLC) cell line, A549, is a leading cause of cancer-related deaths worldwide and is typically an incurable disease [51]. According to Fig.5a, there is no apparent toxicity by any concentration of tPENs, which makes them potentially safe and effective NPs. The MTT assay also revealed that free DOX triggered a significant cytotoxicity effect (65.28 ± 3.98) on the A549 cell line at the highest concentration (400nM), which was significantly increased after loading in tPENs (56.60 ± 2.36, p value of 0.009). As depicted in Fig.5a, 100nM free miR-34a could reduce the viability of the A549 cell line by 20% over 24h (80.39 ± 10.49). However, after co-treatment, a significant impact on the cell viability (52.29 ± 3.26) was observed at concentrations of 400nM DOX and 100nM miR-34a after 24h. These results suggest that the cytotoxicity of DOX/ miR-34a-loaded tPENs was more significant than DOX-loaded tPENs and also the free drugs. After that, the effects of tPENs, DOX, miR-34a, and co-loaded tPENs on the actin cytoskeleton of A549 cells were assessed (Fig.5). Therefore, the treated cells were stained by Alexa fluor 532-labeled phalloidin to show filamentous actin. Actin appeared more equally throughout the cell in tPENs-treated cells (Fig.5b). As expected, DOX induced actin cytoskeleton remodeling with the formation of a cortical contractile ring at the cell periphery and the disruption of central stress fibers (Fig.5c) inconsistent with Wei etal. [52]. In miR-34a treatment, the distribution of the actin cytoskeleton was partially unorganized and tended to form a cortical contractile ring at the cell periphery Fig. 5 Cytotoxicity of tPENs, free drugs (i.e., DOX and miR-34a), and co-loaded tPENs against A549 cell line. The letters show p value greater than 0.05 in every concentration (a). Effects of tPENs (b), DOX (c), miR-34a (d), and co-loaded tPENs (e) on actin cytoskeleton of A549 cells. Images were acquired with × 60 objective, at identical exposure times, and scaled identically. Scale bars are 30μm Vol.:(0123456789) Discover Nano (2023) 18:38 | https://doi.org/10.1186/s11671-023-03781-0 Research 1 3 (Fig.5d). Co-treatment with DOX and miR-34a for 24h also induces cortical actin formation and reduction of stress fiber formation (Fig.5e). Moreover, co-treatment with DOX and miR-34a simultaneously induces the disruption of central stress fibers and increases cortical actin formation. Cellular internalization oftPENs andPENs Targeting, passive or active, significantly impacts the therapeutic effects of NPs [53]. Drug efficacy and safety profiles of NPs can be enhanced by surface modifications in active targeting and physical features in passive targeting (i.e., size, functional groups, charge, and hydrophilicity of the surface) [53]. Herein, Mans has been utilized as a ligand with a high affinity to the Mans receptors as a prospective target for lung cancer therapy [23]. Therefore, the cellular uptake efficiency of labeled PENs and tPENs by FITC was assayed after 2h incubation with A549, which expresses Mans receptors, and NIH-3T3 cell line as a negative control. In confocal microscopy images, the fluorescence of the labeled PENs was exhibited in the cytoplasm of the cells indicating the cellular internalization. According to Fig.6b, c, tPENs showed comparable cell uptake levels to PENs in A549 cells; however, there was no discernible difference between tPENs and PENs in NIH-3T3 cells (Fig.6e, f). In addition, A549 cells demonstrated significantly higher uptake levels than NIH-3T3 cells. Moreover, active targeting could increase cellular uptake by particular ligand-receptor interactions. Conclusion The engineered formulation showed a significantly higher amount of Carr’s index and FPF, and a lower yield in the presence of leucine. Nonetheless, the inclusion of Mannitol was crucial to decrease dG and dA,theory, and increasing yield. MMAD below 6µm confirmed that the formulation could be a suitable drug carrier for treating the TB region, especially in the first generations of branching. NIMs tended to release DOX in a more sustained manner than tPENs Fig. 6 Confocal microscopy images of cellular internalization in untreated (a) and treated A549 cells with FITC-labeled PENs (b) and tPENs (c) in comparison with untreated (d) and treated NIH-3T3 cells by FITC-labeled PENs (e) and tPENs (f) after 2h incubation at 37.0°C Vol:.(1234567890) Research Discover Nano (2023) 18:38 | https://doi.org/10.1186/s11671-023-03781-0 1 3 50. Motiei M, Aboutalebi F, Forouzanfar M, Dormiani K, Nasr-Esfahani MH, Mirahmadi-Zare SZ. Smart co-delivery of miR-34a and cytotoxic peptides (LTX-315 and melittin) by chitosan based polyelectrolyte nanocarriers for specific cancer cell death induction. Mater Sci Eng C. 2021;128: 112258. 51. Perry JL, Tian S, Sengottuvel N, Harrison EB, Gorentla BK, Kapadia CH, etal. Pulmonary delivery of nanoparticle-bound toll-like receptor 9 agonist for the treatment of metastatic lung cancer. ACS Nano. 2020;14(6):7200–15. 52. 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