Structure-Based Evaluation of Hybrid Lipid–Polymer Nanoparticles: The Role of the Polymeric Guest

The combination of phospholipids and block-copolymers yields advanced hybrid nanoparticles through the self-assembly process in an aqueous environment. The physicochemical features of the lipid/polymer components, like the lipid–polymer molar ratio, the macromolecular architecture of the block copolymer, the main transition temperature of the phospholipid, as well as the formulation and preparation protocol parameters, are some of the most crucial parameters for the formation of hybrid lipid/polymer vesicles and for the differentiation of their morphology. The morphology, along with other physicochemical nanoparticle characteristics are strictly correlated with the nanoparticle’s later biological behavior after being administered, affecting interactions with cells, biodistribution, uptake, toxicity, drug release, etc. In the present study, a structural evaluation of hybrid lipid–polymer nanoparticles based on cryo-TEM studies was undertaken. Different kinds of hybrid lipid–polymer nanoparticles were designed and developed using phospholipids and block copolymers with different preparation protocols. The structures obtained ranged from spherical vesicles to rod-shaped structures, worm-like micelles, and irregular morphologies. The obtained morphologies were correlated with the formulation and preparation parameters and especially the type of lipid, the polymeric guest, and their ratio.


Introduction
Drug delivery systems are widely used for the improvement of the pharmacological and pharmacokinetic profiles of active substances.Nanocarriers are drug delivery systems and are composed of different kinds of excipients, i.e., polymers, lipids, surfactants, inorganic materials, etc.Each class of nanocarriers utilized for drug delivery exhibits several advantages and limitations for clinical use.For this reason, the combination of different materials is an attractive methodology for the fast clinical translation of the drug delivery nanocarriers [1][2][3][4].
By combining phospholipids and block copolymers, hybrid nanoparticles can be formed in an aqueous environment.The molar ratio of the components, the molecular weight and macromolecular architecture of the block copolymer, the main transition temperature of the phospholipid, and the surface charge, accompanied by the selection of an appropriate preparation protocol, are the crucial parameters for the formation of hybrid lipid/polymer vesicles and other nanomorphologies [1,[5][6][7][8][9].Furthermore, spheroplexes are fabricated with hybrid PLGA-cationic lipid nanoparticles for in vitro and oral delivery of siRNA [10].Hybrid nanoparticles composed of the biocompatible polymer poly(methyl methacrylate) (PMMA) and the cationic lipid dioctadecyl dimethyl ammonium bromide (DODAB) and produced via the emulsion polymerization of methyl methacrylate (MMA) Polymers 2024, 16, 290 3 of 13
structure-based characterization of hybrid lipid-polymer nanoparticles and for the connection of their structure with the components of the system, especially the polymeric guest.

Preparation of polymer-lipid hybrid nanoparticles
Thin-film hydration methods and aqueous heat methods used as previously described [24, 25,31,32].Briefly, for the aqueous heat method, phospholipids/block copolymer were mixed into HPLC-grade water to a concentration of 2 or 5 mg/mL (colloidal concentration), heated to 70 °C (at least 10 degrees °C higher temperature than the temperature above Tm (main transition temperature) of each lipid) for 30 min with a rate of 5 °C/min for 30 min with intermittent resting periods of 5 min (600 rpm using a magnetic stirrer), and then cooled to room temperature at a rate of 5 °C/min [32].For the thin-film hydration method, desired amounts of phospholipids and block copolymer were dissolved in chloroform/methanol (9:1 v/v) and then transferred into a round flask connected to a rotary evaporator.A vacuum was applied, and the mixed phospholipids/block copolymer thin film was formed by slow removal of the solvent at 45-50 °C.The mixed film was maintained under vacuum for at least 24 h in a desiccator to remove traces of solvent and, subsequently, was hydrated in HPLC-grade water via slow stirring for 1 h in a water bath above the phase transition temperature of lipids.The resultant nanostructures were subjected to two 5 min sonication cycles (amplitude 70; cycle 0.7) interrupted by a 5 min resting period, in a water bath, using a probe sonicator.The resultant nanostructures were allowed to anneal for 30 min [31].

Preparation of Polymer-Lipid Hybrid Nanoparticles
Thin-film hydration methods and aqueous heat methods used as previously described [24, 25,31,32].Briefly, for the aqueous heat method, phospholipids/block copolymer were mixed into HPLC-grade water to a concentration of 2 or 5 mg/mL (colloidal concentration), heated to 70 • C (at least 10 degrees • C higher temperature than the temperature above Tm (main transition temperature) of each lipid) for 30 min with a rate of 5 • C/min for 30 min with intermittent resting periods of 5 min (600 rpm using a magnetic stirrer), and then cooled to room temperature at a rate of 5 • C/min [32].For the thin-film hydration method, desired amounts of phospholipids and block copolymer were dissolved in chloroform/methanol (9:1 v/v) and then transferred into a round flask connected to a rotary evaporator.A vacuum was applied, and the mixed phospholipids/block copolymer thin film was formed by slow removal of the solvent at 45-50 • C. The mixed film was maintained under vacuum for at least 24 h in a desiccator to remove traces of solvent and, subsequently, was hydrated in HPLC-grade water via slow stirring for 1 h in a water bath above the phase transition temperature of lipids.The resultant nanostructures were subjected to two 5 min sonication cycles (amplitude 70; cycle 0.7) interrupted by a 5 min resting period, in a water bath, using a probe sonicator.The resultant nanostructures were allowed to anneal for 30 min [31].

Cryogenic Transmission Electron Microscopy (Cryo-TEM)
Cryogenic Transmission Electron Microscopy (cryo-TEM) images were obtained using a Tecnai F20 X TWIN microscope (FEI Company, Hillsboro, OR, USA) equipped with a field emission gun, operating at an acceleration voltage of 200 kV.Images were recorded on a Gatan Rio 16 CMOS 4k camera (Gatan Inc., Pleasanton, CA, USA) and processed with Gatan Microscopy Suite (GMS) software version 3.31.2360.0(Gatan Inc., Pleasanton, CA, USA).Specimen preparation was performed via vitrification of the aqueous solutions on grids with holey carbon film (Quantifoil R 2/2; Quantifoil Micro Tools GmbH, Großlöbichau, Germany).Prior to use, the grids were activated for 15 s in oxygen plasma using a Femto plasma cleaner (Diener Electronic, Ebhausen, Germany).Cryo-samples were prepared by applying a droplet (3 µL) of the suspension to the grid, blotting with filter paper and immediate freezing in liquid ethane using a fully automated blotting device Vitrobot Mark IV (Thermo Fisher Scientific, Waltham, MA, USA).After preparation, the vitrified specimens were kept under liquid nitrogen until they were inserted into a cryo-TEM-holder Gatan 626 (Gatan Inc., Pleasanton, CA, USA) and analyzed in the TEM at −178 • C.

Dynamic and Electrophoretic Light Scattering
The physicochemical behavior of the prepared nanosystems was characterized by measuring the size (hydrodynamic radius R h , nm) and the size distribution (polydispersity index, PDI) using dynamic light scattering (DLS), while the ζ-potential (ζ-pot, mV) of the nanoparticles was measured by electrophoretic light scattering (ELS).In total, 100 µL of aliquots was 30-fold diluted in HPLC-grade water.Dynamic light scattering measurements were performed using an AVL/CGS-3 Compact Goniometer System (ALV GmbH, Berlin, Germany), equipped with a cylindrical JDS Uniphase 22 mV He-Ne laser, operating at 632.8 nm, and an Avalanche photodiode detector.The system was interfaced with an ALV/LSE-5003 electronics unit for the stepper motor drive and limit switch control and an ALV-5000/EPP multi-tau digital correlator.Autocorrelation functions were analyzed using the cumulants method and the CONTIN.Apparent hydrodynamic radii, R h , was calculated at finite concentrations using the Stokes-Einstein equation: where k B is the Boltzmann constant, n 0 is the viscosity of water at temperature T, and D is the diffusion coefficient at a fixed concentration.The polydispersity of the particle sizes was given as the µ 2 /Γ 2 (PDI) from the cumulants method, where Γ is the average relaxation rate, and µ 2 is its second moment.For the ELS, 100 µL of aliquots was 30-fold diluted in HPLC-grade water, and ζ-potential was measured at room temperature at 633 nm.The ζ-potentials were calculated from electrophoretic mobilities using the Henry correction of the Smoluchowski equation.More details regarding the DLS [24] and ELS [24,31] protocols are described in detail in our previous studies.The R h , PDI, and ζ-pot values of the nanoparticles were averaged from triplicate measurements, and the results were reported as a mean ± standard deviation.

Hybrid Nanoparticles Employing POEGMA-PLA
The DPPC/HSPC:POEGMA-PLA polymer-lipid hybrid nanoparticles were prepared using the thin-film hydration method followed by probe sonication as a size reduction method.In both cases of hybrid nanoparticles with POEGMA-PLA, the size of these polymer-lipid hybrid nanoparticles was at the nanoscale (below 100 nm) and the size distribution was as narrow as the PDI values indicated (Table 1).
The presence of HSPC led to a slightly higher hydrodynamic radius (R h ) of the nanoparticles, probably due to the higher main transition temperature (Tm) of this phospholipid in comparison to DPPC.The Tm is considered a key parameter for the preparation of lipid-based nanoparticles because it governs the self-assembly process during the hydration of the thin film.The cryo-TEM images showed nanoparticles that were mainly vesicular and unilamellar, with inhomogeneous incorporation of the POEMA-PLA copolymer into the lipid bilayer (Figure 2).The presence of HSPC led to a slightly higher hydrodynamic radius (Rh) of the nanoparticles, probably due to the higher main transition temperature (Tm) of this phospholipid in comparison to DPPC.The Tm is considered a key parameter for the preparation of lipid-based nanoparticles because it governs the self-assembly process during the hydration of the thin film.The cryo-TEM images showed nanoparticles that were mainly vesicular and unilamellar, with inhomogeneous incorporation of the POEMA-PLA copolymer into the lipid bilayer (Figure 2).The size of the polymer-lipid hybrid nanoparticles is almost the same as the values observed by DLS (Table 1).The thickness of the hybrid membrane was found to be between 6 and 8 nm, which in all cases was higher in comparison to pure liposomal membranes [33].In particular, in the case of DPPC:POEGMA-PLA 9:0.2 (Figure 2a), cryo-TEM revealed spherical or irregular-shaped particles with sizes in the range of 30-150 nm and a membrane thickness of ~6 nm [33].The HSPC:POEGMA-PLA polymer-lipid hybrid nanoparticles exhibited a larger size and an inhomogeneous distribution in comparison to the DPPPC:POEGMA-PLA.In other words, replacement DPPC with HSPC leads to the formation of spherical or irregular-shaped particles (Figure 2b) with sizes in the range of 40-215 nm and a membrane thickness of ~6 nm.The latter means that the selection of the lipid is important for altering the morphological and physical properties of polymer-lipid hybrid nanoparticles.The size distribution is presented in Figure 3, and stability study over time is presented in Figure S1, confirming their colloidal stability.Histograms were The size of the polymer-lipid hybrid nanoparticles is almost the same as the values observed by DLS (Table 1).The thickness of the hybrid membrane was found to be between 6 and 8 nm, which in all cases was higher in comparison to pure liposomal membranes [33].In particular, in the case of DPPC:POEGMA-PLA 9:0.2 (Figure 2a), cryo-TEM revealed spherical or irregular-shaped particles with sizes in the range of 30-150 nm and a membrane thickness of ~6 nm [33].The HSPC:POEGMA-PLA polymer-lipid hybrid nanoparticles exhibited a larger size and an inhomogeneous distribution in comparison to the DPPPC:POEGMA-PLA.In other words, replacement DPPC with HSPC leads to the formation of spherical or irregular-shaped particles (Figure 2b) with sizes in the range of 40-215 nm and a membrane thickness of ~6 nm.The latter means that the selection of the lipid is important for altering the morphological and physical properties of polymer-lipid hybrid nanoparticles.The size distribution is presented in Figure 3, and stability study over time is presented in Figure S1, confirming their colloidal stability.Histograms were prepared by measuring 100 particles from cryo-TEM images.The number of particles in the histograms depends on the particle concentration.Generally, the more particles measured, the higher the accuracy of the histograms.We tried to measure at least 100 particles, although this is not always possible due to their low concentration in the sample.Pure polymeric nanostructures of POEGMA-PLA in the form of micelles were prepared in water and exhibited a mean hydrodynamic diameter of 32 nm with PDI at 0.21, thus being smaller in comparison to the mixed nanostructures with lipid.
the histograms depends on the particle concentration.Generally, the more particles measured, the higher the accuracy of the histograms.We tried to measure at least 100 particles, although this is not always possible due to their low concentration in the sample.Pure polymeric nanostructures of POEGMA-PLA in the form of micelles were prepared in water and exhibited a mean hydrodynamic diameter of 32 nm with PDI at 0.21, thus being smaller in comparison to the mixed nanostructures with lipid.

Hybrid Nanoparticles Employing PLMA-b-PDMAEMA
The aqueous-heat method was used for the preparation of DSPC:DAP:PLMA-b-PDMAEMA nanosystems at 1:0.7:0.03 and 1:1:0.03weight ratios, without any other method for the size reduction of the systems.The colloidal concentration was equal to 2 mg/ml.In this case, we kept the weight ratio stable for the zwitterionic lipid DSPC/polymer and increased the weight ratio for the cationic lipid DAP.The increase in DAP led to a more homogenous population of particles with a smaller size and higher positive (as expected) values of zeta potential.The DSPC:DAP:PLMA-b-PDMAEMA 1:1:0.03weight ratio polymer-lipid hybrid nanoparticles were also at the nanoscale.The last observation is quite important because we achieved these physicochemical characteristics without using any size reduction method.The cryo-TEM images showed two kinds of species: unilamellar-vesicular and rod-like structures.
In the case of DSPC:DAP:PLMA-b-PDMAEMA 1:0.7:0.03wt ratio (Figure 4a), cryo-TEM revealed the predominant spherical or irregular-shaped particles with sizes in the range of 30-410 nm.As shown in Figure 4, there is a significant difference in the particle sizes ranging from 100 nm to 400 nm for DSPC:DAP:PLMA-b-PDMAEMA 1:0.7:0.03weight ratio and between 100 nm and 1000 nm for DSPC:DAP:PLMA-b-PDMAEMA 1:1:0.03weight ratio polymer-lipid hybrid nanoparticles.On the other hand, as shown in Figure 5, the main population of the nanoparticles exhibits a size of around 100 nm.This phenomenon is strongly associated with the preparation protocol of hybrid particles.As mentioned before, we did not use any size reduction method (i.e., extrusion through polycarbonate filters) in order to achieve extremely low polydispersity.We assume that the obtained high number of the nanoparticles is multifactorial, being due to both the preparation method and the presence of the helper lipid DAP that, acting like a surfactant, increases the inner surface by crumbling the large nanoparticles into smaller ones of a higher population.The self-assembly of these materials is the reason for the size and morphology heterogeneity.The size distribution is presented in Figure 5, and stability study over time is presented in Figure S2, confirming their colloidal stability.Histograms were prepared by measuring 200 particles from cryo-TEM images.There were also rod-like particles observed as a minority component.For DSPC:DAP:PLMA-b-PDMAEMA 1:1:0.03wt ratio nanosystems, cryo-TEM revealed the predominant spherical or irregular-shaped particles

Hybrid Nanoparticles Employing PLMA-b-PDMAEMA
The aqueous-heat method was used for the preparation of DSPC:DAP:PLMA-b-PDMAEMA nanosystems at 1:0.7:0.03 and 1:1:0.03weight ratios, without any other method for the size reduction of the systems.The colloidal concentration was equal to 2 mg/mL.In this case, we kept the weight ratio stable for the zwitterionic lipid DSPC/polymer and increased the weight ratio for the cationic lipid DAP.The increase in DAP led to a more homogenous population of particles with a smaller size and higher positive (as expected) values of zeta potential.The DSPC:DAP:PLMA-b-PDMAEMA 1:1:0.03weight ratio polymer-lipid hybrid nanoparticles were also at the nanoscale.The last observation is quite important because we achieved these physicochemical characteristics without using any size reduction method.The cryo-TEM images showed two kinds of species: unilamellar-vesicular and rod-like structures.
In the case of DSPC:DAP:PLMA-b-PDMAEMA 1:0.7:0.03wt ratio (Figure 4a), cryo-TEM revealed the predominant spherical or irregular-shaped particles with sizes in the range of 30-410 nm.As shown in Figure 4, there is a significant difference in the particle sizes ranging from 100 nm to 400 nm for DSPC:DAP:PLMA-b-PDMAEMA 1:0.7:0.03weight ratio and between 100 nm and 1000 nm for DSPC:DAP:PLMA-b-PDMAEMA 1:1:0.03weight ratio polymer-lipid hybrid nanoparticles.On the other hand, as shown in Figure 5, the main population of the nanoparticles exhibits a size of around 100 nm.This phenomenon is strongly associated with the preparation protocol of hybrid particles.As mentioned before, we did not use any size reduction method (i.e., extrusion through polycarbonate filters) in order to achieve extremely low polydispersity.We assume that the obtained high number of the nanoparticles is multifactorial, being due to both the preparation method and the presence of the helper lipid DAP that, acting like a surfactant, increases the inner surface by crumbling the large nanoparticles into smaller ones of a higher population.The self-assembly of these materials is the reason for the size and morphology heterogeneity.The size distribution is presented in Figure 5, and stability study over time is presented in Figure S2, confirming their colloidal stability.Histograms were prepared by measuring 200 particles from cryo-TEM images.There were also rod-like particles observed as a minority component.For DSPC:DAP:PLMA-b-PDMAEMA 1:1:0.03wt ratio nanosystems, cryo-TEM revealed the predominant spherical or irregular-shaped particles with sizes in the range of 30-300 nm.There was also a small number of particles larger than 300 nm.The size distribution is presented in Figure 5b.The particles were co-existing with rod-like particles.At the lowest weight ratio of DAP, the presence of rod-like structures was observed, and these structures exhibited lengths of around 100 nm.
with sizes in the range of 30-300 nm.There was also a small number of particles larger than 300 nm.The size distribution is presented in Figure 5b.The particles were co-existing with rod-like particles.At the lowest weight ratio of DAP, the presence of rod-like structures was observed, and these structures exhibited lengths of around 100 nm.According to the literature, the elongated shaped morphologies, like the present rodlike structures, are correlated with an increase in the circulation time due to their elongated form, which performs well in flow and has been shown to prevent phagocytosis through flow-induced shear forces [34].According to the literature, the aspect ratio of rodlike nanoparticles affects cellular internalization, with particle length having a key role in differentiating the biological pathways taking place.More specifically, the rod-shaped particles have been found to exhibit greater transport across intestinal cells compared to their spherical counterparts, enhancing their accumulation in the desired cells, and thus being ideal for oral drug delivery applications where intestine penetration is crucial for bioavailability enhancement [35].Toxicity issues due to morphology are also raised.According to the literature, biodistribution studies have highlighted that particle morphology not only affects behavior in the bloodstream but also the localization in various tissues, with elongated particles showing the least accumulation in the kidneys [27].Thus, we can expect that morphology, being dependent on the lipid-polymer ratio, can also affect the biological behavior of the nanosystems, the nanoparticle-biological system interactions, and their pharmacokinetic and excretion profile.with sizes in the range of 30-300 nm.There was also a small number of particles larger than 300 nm.The size distribution is presented in Figure 5b.The particles were co-existing with rod-like particles.At the lowest weight ratio of DAP, the presence of rod-like structures was observed, and these structures exhibited lengths of around 100 nm.
(a) (b)  According to the literature, the elongated shaped morphologies, like the present rodlike structures, are correlated with an increase in the circulation time due to their elongated form, which performs well in flow and has been shown to prevent phagocytosis through flow-induced shear forces [34].According to the literature, the aspect ratio of rodlike nanoparticles affects cellular internalization, with particle length having a key role in differentiating the biological pathways taking place.More specifically, the rod-shaped particles have been found to exhibit greater transport across intestinal cells compared to their spherical counterparts, enhancing their accumulation in the desired cells, and thus being ideal for oral drug delivery applications where intestine penetration is crucial for bioavailability enhancement [35].Toxicity issues due to morphology are also raised.According to the literature, biodistribution studies have highlighted that particle morphology not only affects behavior in the bloodstream but also the localization in various tissues, with elongated particles showing the least accumulation in the kidneys [27].Thus, we can expect that morphology, being dependent on the lipid-polymer ratio, can also affect the biological behavior of the nanosystems, the nanoparticle-biological system interactions, and their pharmacokinetic and excretion profile.According to the literature, the elongated shaped morphologies, like the present rodlike structures, are correlated with an increase in the circulation time due to their elongated form, which performs well in flow and has been shown to prevent phagocytosis through flow-induced shear forces [34].According to the literature, the aspect ratio of rod-like nanoparticles affects cellular internalization, with particle length having a key role in differentiating the biological pathways taking place.More specifically, the rod-shaped particles have been found to exhibit greater transport across intestinal cells compared to their spherical counterparts, enhancing their accumulation in the desired cells, and thus being ideal for oral drug delivery applications where intestine penetration is crucial for bioavailability enhancement [35].Toxicity issues due to morphology are also raised.According to the literature, biodistribution studies have highlighted that particle morphology not only affects behavior in the bloodstream but also the localization in various tissues, with elongated particles showing the least accumulation in the kidneys [27].Thus, we can expect that morphology, being dependent on the lipid-polymer ratio, can also affect the biological behavior of the nanosystems, the nanoparticle-biological system interactions, and their pharmacokinetic and excretion profile.
The presence of cationic lipid seems to play a key role in the formation of other structures, except spherical ones.Previously, in lower concentrations of DAP, we also observed "spaghetti" structures, which exhibited different physicochemical characteristics [36].The same structures have also appeared in complex formations between cationic liposomes and nucleic acids [36].
Taking into consideration the already existing literature [37], the introduction of the cationic lipid DAP in the lipidic formulation consisting only of DSPC and PLMA-b-PDMAEMA copolymer led to an increase in the grade of organization from spherical or irregular particles with low contrast, with a size range of 50-600 nm, to the present structures of more homogenous morphology and smaller sizes.In our previous work [37], we concluded that an amount of copolymer PLMA-b-PDMAEMA is not fully incorporated in the DSPC formulations, which can contribute to "threadlike" structures that cover the vesicles.In the present work, we observe that the presence of DAP can assist in the better incorporation of the copolymer, transforming the "threadlike" structures into a coexistence of spherical and rod-like particles.
We also checked the physicochemical characteristics of polymer-lipid hybrid nanoparticles composed of PDMAEMA-b-PLMA without the presence of cationic lipid.We used zwitterionic lipids with different T m , i.e., DMPC with T m at 24 • C and DPPC with Tm at 41 • C. The aqueous heat method was also used as a preparation protocol, and the colloidal concentration was equal to 5 mg/mL.We observed the same trend that we described previously.In particular, the presence of DPPC led to a higher hydrodynamic radius (R h ) of the nanoparticles and higher PDI values, probably due to the higher main transition temperature (T m ) of this phospholipid in comparison to DMPC.So, the Tm of the phospholipid seems to be another crucial parameter for the design of hybrid polymer lipid nanoparticles.In contrast, the cryo-TEM images showed the formation of DMPC-based structures with irregular shapes with sizes of around 600 nm, except for the vesicles with a thickness of the hybrid membrane being in the range of 8-10 nm.The DPPC:PDMAEMA-b-PLMA 95:5 weight ratio polymer-lipid hybrid nanoparticles were found to be between 150 and 300 nm in diameter with spherical or polygonal shapes (Figure 6).
The presence of cationic lipid seems to play a key role in the formation of other structures, except spherical ones.Previously, in lower concentrations of DAP, we also observed "spaghetti" structures, which exhibited different physicochemical characteristics [36].The same structures have also appeared in complex formations between cationic liposomes and nucleic acids [36].
Taking into consideration the already existing literature [37], the introduction of the cationic lipid DAP in the lipidic formulation consisting only of DSPC and PLMA-b-PDMAEMA copolymer led to an increase in the grade of organization from spherical or irregular particles with low contrast, with a size range of 50-600 nm, to the present structures of more homogenous morphology and smaller sizes.In our previous work [37], we concluded that an amount of copolymer PLMA-b-PDMAEMA is not fully incorporated in the DSPC formulations, which can contribute to "threadlike" structures that cover the vesicles.In the present work, we observe that the presence of DAP can assist in the better incorporation of the copolymer, transforming the "threadlike" structures into a coexistence of spherical and rod-like particles.
We also checked the physicochemical characteristics of polymer-lipid hybrid nanoparticles composed of PDMAEMA-b-PLMA without the presence of cationic lipid.We used zwitterionic lipids with different Tm, i.e., DMPC with Tm at 24 °C and DPPC with Tm at 41 °C.The aqueous heat method was also used as a preparation protocol, and the colloidal concentration was equal to 5 mg/mL.We observed the same trend that we described previously.In particular, the presence of DPPC led to a higher hydrodynamic radius (Rh) of the nanoparticles and higher PDI values, probably due to the higher main transition temperature (Tm) of this phospholipid in comparison to DMPC.So, the Tm of the phospholipid seems to be another crucial parameter for the design of hybrid polymer lipid nanoparticles.In contrast, the cryo-TEM images showed the formation of DMPC-based structures with irregular shapes with sizes of around 600 nm, except for the vesicles with a thickness of the hybrid membrane being in the range of 8-10 nm.The DPPC:PDMAEMA-b-PLMA 95:5 weight ratio polymer-lipid hybrid nanoparticles were found to be between 150 and 300 nm in diameter with spherical or polygonal shapes (Figure 6).In the case of DMPC:PDMAEMA-b-PLMA 95:5 wt ratio (Figure 6a), cryo-TEM revealed the predominant spherical or irregular-shaped particles with sizes in the range of 50-300 nm.The particles have a distinct membrane with a thickness of 7-10 nm.The size distribution is presented in Figure 7a, and stability study over time is presented in Figure S3, confirming their colloidal stability.Histograms were prepared by measuring 200 particles from cryo-TEM images.This large population coexisted with oval or irregularshaped particles with a striped structure and sizes in the range of 100-600.In the case of DMPC:PDMAEMA-b-PLMA 95:5 wt ratio (Figure 6a), cryo-TEM revealed the predominant spherical or irregular-shaped particles with sizes in the range of 50-300 nm.The particles have a distinct membrane with a thickness of 7-10 nm.The size distribution is presented in Figure 7a, and stability study over time is presented in Figure S3, confirming their colloidal stability.Histograms were prepared by measuring 200 particles from cryo-TEM images.This large population coexisted with oval or irregular-shaped particles with a striped structure and sizes in the range of 100-600.From a biological point of view, this morphological diversity of the nanovesicles, being either spherical or irregular-shaped or polygonal-shaped particles, can be a projection of their later biodistribution behavior.According to the literature, the larger morphological diversity is considered to demonstrate a higher specific adhesion and more limited non-specific interactions, while being in the bloodstream, over the more rigid, spherical ones, due to their propensity to adhere to the vascular wall.Moreover, an increase in the circulation time of elongated particles is expected because they can perform well in flow, while phagocytosis is also prevented through flow-induced shear forces [27,34].
Furthermore, it is very interesting, from a physicochemical point of view, to focus a little more on the observed striped elongated morphological pattern of some nanoassemblies.This unique striped pattern of the nanoparticles at the DMPC:PDMAEMA-b-PLMA 95:5 (Figure 6a) ratio resembles images from polymeric poly(ionic liquid) (PIL) nanoparticles [38][39][40], where, independent of nanoparticle size, ellipsoids with interior alternating latitudinal stripes, that is, a "wasp-like" pattern with parallel lamellae, were found.The light and dark lamellae stem from the hydrophobic alkyl chains of both the polymer and the phospholipid chains and the charged polymer backbones, respectively, due to higher electron density compared to that of the carbon atoms.According to Zhang et al. [39] and Klinger et al. [41], with increasing hydrophobicity in longer alkyl chains, their exposure at the surface is energetically unfavorable, and the intraparticle morphologies shift from ellipsoid (wasp-like) to spherical ones, a result of the energy balance of interfacial energy between the hydrophobic alkyl chains and the hydrophilic charged backbones and the surface energy of the particle.This phenomenon can explain the morphological transformation that we observe when the lipid DMPC of 14 carbon alkyl chains is replaced by the lipid DPPC of 16 carbon alkyl chains.
On the other hand, different solid DPPC polymorphs, called ripple and tilt (or gel), are hypothesized to be the source of the patch-and striped-shaped domains, showing that the lower-melting phospholipid can be replaced by an appropriate copolymer to improve mechanical properties while preserving the underlying membrane physics in mixed-phospholipid bilayers when a high-melting phospholipid solidifies on cooling [42].Apart from this category of particles, there were also rod-like particles observed as a minority component [43].The replacement of DMPC with DPPC, in prepared hybrids, leads to a different, prevailing morphology of nanosystems (Figure 6b).Predominantly spherical particles were observed with sizes in the range of 50-450 nm and a membrane thickness of 7-8 nm [43].The size distribution is presented in Figure 7b.This large population coexisted with pentagonal-shaped particles with a diagonal length of 60-100 nm and a wall thickness of ~8 nm.Apart from this category of particles, there were also rod-like particles observed as a minority component.
With a complete picture of the physicochemical and morphological characteristics of polymer-lipid hybrid nanoparticles composed of the amphiphilic block copolymer PLMA-b-PDMAEMA and phospholipids that are widely used for drug delivery purposes, we chose lipids that are widely used for vaccine delivery with adjuvant properties.DDA From a biological point of view, this morphological diversity of the nanovesicles, being either spherical or irregular-shaped or polygonal-shaped particles, can be a projection of their later biodistribution behavior.According to the literature, the larger morphological diversity is considered to demonstrate a higher specific adhesion and more limited nonspecific interactions, while being in the bloodstream, over the more rigid, spherical ones, due to their propensity to adhere to the vascular wall.Moreover, an increase in the circulation time of elongated particles is expected because they can perform well in flow, while phagocytosis is also prevented through flow-induced shear forces [27,34].
Furthermore, it is very interesting, from a physicochemical point of view, to focus a little more on the observed striped elongated morphological pattern of some nanoassemblies.This unique striped pattern of the nanoparticles at the DMPC:PDMAEMA-b-PLMA 95:5 (Figure 6a) ratio resembles images from polymeric poly(ionic liquid) (PIL) nanoparticles [38][39][40], where, independent of nanoparticle size, ellipsoids with interior alternating latitudinal stripes, that is, a "wasp-like" pattern with parallel lamellae, were found.The light and dark lamellae stem from the hydrophobic alkyl chains of both the polymer and the phospholipid chains and the charged polymer backbones, respectively, due to higher electron density compared to that of the carbon atoms.According to Zhang et al. [39] and Klinger et al. [41], with increasing hydrophobicity in longer alkyl chains, their exposure at the surface is energetically unfavorable, and the intraparticle morphologies shift from ellipsoid (wasp-like) to spherical ones, a result of the energy balance of interfacial energy between the hydrophobic alkyl chains and the hydrophilic charged backbones and the surface energy of the particle.This phenomenon can explain the morphological transformation that we observe when the lipid DMPC of 14 carbon alkyl chains is replaced by the lipid DPPC of 16 carbon alkyl chains.
On the other hand, different solid DPPC polymorphs, called ripple and tilt (or gel), are hypothesized to be the source of the patch-and striped-shaped domains, showing that the lower-melting phospholipid can be replaced by an appropriate copolymer to improve mechanical properties while preserving the underlying membrane physics in mixed-phospholipid bilayers when a high-melting phospholipid solidifies on cooling [42].Apart from this category of particles, there were also rod-like particles observed as a minority component [43].The replacement of DMPC with DPPC, in prepared hybrids, leads to a different, prevailing morphology of nanosystems (Figure 6b).Predominantly spherical particles were observed with sizes in the range of 50-450 nm and a membrane thickness of 7-8 nm [43].The size distribution is presented in Figure 7b.This large population coexisted with pentagonal-shaped particles with a diagonal length of 60-100 nm and a wall thickness of ~8 nm.Apart from this category of particles, there were also rod-like particles observed as a minority component.
With a complete picture of the physicochemical and morphological characteristics of polymer-lipid hybrid nanoparticles composed of the amphiphilic block copolymer PLMAb-PDMAEMA and phospholipids that are widely used for drug delivery purposes, we chose lipids that are widely used for vaccine delivery with adjuvant properties.DDA and TBD are well-established in the literature for their properties in the design and development of subunit vaccines.In this case, we kept the weight ratio of the lipids constant and increased the weight ratio of the polymeric guest with fixed colloidal concentration to 2 mg/mL.The aqueous heat method was used for the formation of the particles without any additional processes for size reduction.The presence of the amphiphilic block copolymer PLMAb-PDMAEMA at a higher weight ratio led to a decrease in the size of the nanoparticles and a higher homogeneity of the population of the particles.For both systems, the zeta potential values were extremely positive, but we should highlight that the presence of PLMA-b-PDMAEMA seems to cover a small percentage of the positive charge of the lipids, and as the copolymer content increased, the zeta potential values decreased.
The cryo-TEM images showed spherical and vesicular structures with sizes almost the same as those measured from DLS experiments, taking into account the polydispersity of the systems [44].In the recent literature, the giant hybrid unilamellar vesicles, as we observed for our systems, have also been called hybridosomes [44].For DDA:TDB:PLMAb-PDMAEMA 1:0.2:1 nanosystems (Figure 8a), cryo-TEM revealed spherical vesicles and irregular-shaped vesicles with sizes in the range of 70-590 nm and a membrane thickness of ~6 nm.For DDA:TDB:PLMA-b-PDMAEMA 1:0.2:2.5 vesicles covered with "threadlike" structures (Figure 8b), sizes in the range of 50-460 nm and a membrane thickness of ~6 nm were observed.This population coexisted with spherical "solid" particles (Figure 8c) with sizes in the range of 20-50 nm, which were sometimes joined together [45].The concentration of particles in both cases was too low to prepare histograms because the overall colloidal concentration was low, while their study over time is presented in Figure S4, confirming their kinetic stability.
Polymers 2024, 16, x FOR PEER REVIEW 10 of 13 and TBD are well-established in the literature for their properties in the design and development of subunit vaccines.In this case, we kept the weight ratio of the lipids constant and increased the weight ratio of the polymeric guest with fixed colloidal concentration to 2 mg/mL.The aqueous heat method was used for the formation of the particles without any additional processes for size reduction.The presence of the amphiphilic block copolymer PLMA-b-PDMAEMA at a higher weight ratio led to a decrease in the size of the nanoparticles and a higher homogeneity of the population of the particles.For both systems, the zeta potential values were extremely positive, but we should highlight that the presence of PLMA-b-PDMAEMA seems to cover a small percentage of the positive charge of the lipids, and as the copolymer content increased, the zeta potential values decreased.The cryo-TEM images showed spherical and vesicular structures with sizes almost the same as those measured from DLS experiments, taking into account the polydispersity of the systems [44].In the recent literature, the giant hybrid unilamellar vesicles, as we observed for our systems, have also been called hybridosomes [44].For DDA:TDB:PLMA-b-PDMAEMA 1:0.2:1 nanosystems (Figure 8a), cryo-TEM revealed spherical vesicles and irregular-shaped vesicles with sizes in the range of 70-590 nm and a membrane thickness of ~6 nm.For DDA:TDB:PLMA-b-PDMAEMA 1:0.2:2.5 vesicles covered with "threadlike" structures (Figure 8b), sizes in the range of 50-460 nm and a membrane thickness of ~6 nm were observed.This population coexisted with spherical "solid" particles (Figure 8c) with sizes in the range of 20-50 nm, which were sometimes joined together [45].The concentration of particles in both cases was too low to prepare histograms because the overall colloidal concentration was low, while their study over time is presented in Figure S4, confirming their kinetic stability.
Taking into account the physicochemical results of our previous study [30], where pure polymeric nanostructures of PDMAEMA-b-PLMA in the form of micelles were studied, the pure micelles exhibited a mean hydrodynamic diameter of 88 nm with PDI at 0.04, being larger in comparison to mixed nanostructures with lipid, except for the two formulations with the combination of DDA and TDB lipids.

Figure 2 .
Figure 2. Cryo-TEM images of (a) DPPC:POEGMA-PLA (9:0.2 weight ratio) and (b) HSPC:POEGMA-PLA (9:0.2 weight ratio) polymer-lipid hybrid nanoparticles.The images for all systems were taken at the same point in time, immediately after preparation for comparison reasons.

Figure 2 .
Figure 2. Cryo-TEM images of (a) DPPC:POEGMA-PLA (9:0.2 weight ratio) and (b) HSPC:POEGMA-PLA (9:0.2 weight ratio) polymer-lipid hybrid nanoparticles.The images for all systems were taken at the same point in time, immediately after preparation for comparison reasons.

Figure 8 .
Figure 8. Cryo-TEM images of DDA:TDB:PLMA-b-PDMAEMA polymer-lipid hybrid nanoparticles at (a) 1:0.2:1 and (b,c) 1:0.2:2.5 wt ratios.The images for all systems were taken at the same point in time, immediately after preparation for comparison reasons.

Table 1 .
The physicochemical characteristics of the polymer-lipid hybrid nanoparticles.