Tailor-made poly(amidoamine)s for controlled complexation and condensation of DNA

A set of polymer carriers for DNA delivery was synthesized by combining monodisperse, sequence-defined poly(amidoamine) (PAA) segments with poly(ethylene oxide) (PEO) blocks. The precise definition of the PAA segments provides the possibility of correlating the chemical structure (monomer sequence) with the resulting biological properties. Three different PAA-PEO conjugates were synthesized by solid-phase supported synthesis, and the cationic nature of the PAA segments was systematically varied. This allows for the tailoring of interactions with double-stranded plasmid DNA (dsDNA). The potential of the PAA-PEO conjugates as non-viral vectors for gene delivery is demonstrated by investigating the dsDNA complexation and condensation properties. Depending on the applied carrier, a transition in polyplex (polymer-DNA ion complex) structures is observed. This reaches from extended ring-like structures to highly compact toroidal structures, where supercoiling of the DNA is induced. An aggregation model is proposed that is based on structural investigations of the polyplexes with atomic force microscopy (AFM) and dynamic light scattering (DLS). While the cationic PAA segment mediates primarily the contact of the carrier to the dsDNA, the PEO block stabilizes the polyplex and generates a "stealth" aggregate, as was suggested by Zeta potentials that were close to zero. The controlled aggregation leads to stable, single-plasmid complexes, and stabilizes the DNA structure itself. This is shown by ethidium bromide intercalation assays and DNase digestion assays. The presented PAA-PEO systems allow for the formation of well-defined single-plasmid polyplexes, preventing hard DNA compression and strongly polydisperse polyplexes. Moreover carrier polymers and the resulting polyplexes exhibit no cytotoxicity, as was shown by viability tests; this makes the carriers potentially suitable for in vivo delivery applications.     

聚乙二醇嵌段树枝状聚赖氨酸共聚物的合成及其基因载体研究

采用液相多肽合成法制备得到窄分子量分布、结构可控的生物相容性聚乙二醇嵌段共聚树枝状聚赖氨酸阳离子功能大分子(PEG-b-Dendritic PLL). 运用¹H NMR核磁共振、凝胶电泳以及荧光淬灭滴定手段对所得阳离子两嵌段大分子的化学结构及其与质粒DNA (pDNA)结合作用与复合行为进行了研究. 结果表明聚乙二醇嵌段树枝状聚赖氨酸与pDNA分子可以在缓冲溶液中形成稳定的胶束, pDNA与阳离子树枝赖氨酸嵌段通过静电相互作用形成胶束核, 其水溶性聚乙二醇嵌段形成水溶性胶束壳, 提高了阳离子大分子/pDNA复合胶束的稳定性. 同时发现随着阳离子嵌段树枝状赖氨酸代数的增加, 阳离子两嵌段大分子与pDNA的结合作用增强, 有利于其作为基因转染生物功能载体的应用.     

Cationic liposomes modified with non-ionic surfactants as effective non-viral carrier for gene transfer

A defined change in formulation components affects the physical and chemical characteristics of cationic liposomes (CLs) carriers in many ways. Therefore, a great degree of control can be exercised over the structure by modifying the CLs with various materials, leading to new innovations for carrier improvement. In the present study, surface modifications of cationic liposomes with non-ionic surfactants—sorbitan monoesters serials (Span 85, 80, 40 and 20) were carried out for developing a new gene transfer carrier. Span modified cationic liposomes (Sp-CLs) were prepared by reverse phase evaporation method (RPV) and self-assemble complexes of antisense oligonucleotides/surfactant modifying cationic liposomes were prepared by auto-coacervation through electrostatic effect. Characterization of Sp-CLs and the self-assembled complex was performed by electron microscope, particle size, zeta potential, turbidity and agarose electrophoresis. Furthermore, in vitro cellular uptake experiment showed that Span plays a role in enhancing the cellular uptake of encapsulated oligonucleotides mediated by Sp-CLs by the endocytosis-dependent route. CLs modified with Span 40 significantly facilitated the cellular uptake by COS-7 cells and HeLa cells; also showed some positive effect on gene expression. That suggests it is a potential non-viral carrier for efficient gene transfer.     

Poly(L‐histidine) with several aminoethyl groups for a new pH‐sensitive DNA carrier

In this study, poly(L ‐histidine) with several aminoethyl groups, i.e. aminated poly(L ‐histidine), is reported to be able to make complexes with DNA and to transfect cells in vitro in the presence of serum. The present study was performed to determine whether the pH of the medium had an influence on the complex formation with DNA, on the cell membrane fusion activity and on the transfection efficiency. Agarose gel retardation assays proved that the polyion complex formation of the aminated poly(L ‐histidine) with DNA was affected by pH of the medium, owing to the basicity (protonation–deprotonation) of the imidazole groups with a pK_a value around 6.0. Hemolysis assay showed that the resulting DNA complex enhanced membrane disruptive ability at endosomal pH. The aminated poly(L ‐histidine) gene carrier demonstrated significant transfection efficacy which was decreased by the inclusion of chloroquine as an endosomolytic agent. These results suggest that the aminated poly(L ‐histidine) promises to be a new pH‐sensitive DNA carrier for endosomal escape. Copyright © 2005 John Wiley & Sons, Ltd.     

Construction of Bovine Serum Albumin/AIE‐Based Quaternary Complexes for Efficient Gene Transfection

High transfection efficiency and superior cell imaging are required for cationic polymers‐based gene delivery system to afford high therapeutic effect but its high toxicity and unstable cell imaging are easily ignored. In this study, cationic amino poly(glycerol methacrylate) derivative (PGMA‐EDA) is used to incorporate bovine serum albumin (BSA) and aggregation‐induced emission (AIE) molecular (tetraphenylethylene derivatives, TPE) as an efficient carrier for gene transfection and intracellular imaging. The obtained polymer/pDNA‐TPE/BSA (PDTB) quaternary nanoparticles (NPs) not only exhibit efficient gene transfection but also show excellent biocompatibility. After inclusion of TPE/BSA (TB) NPs, BSA promoted dissociation of the complexes upon being protonated and the lipophilic TPE‐reduced endosomal membrane stability, which enhanced endosomal escape of pDNA payload, finally resulting in an excellent gene transfection. On the other hand, less positive surface charge of PDTB NPs than that of the binary PD complexes, as well as the addition of biocompatible BSA, both factors contribute to the improved cell viability. Moreover, the AIE feature of TPE compared to aggregation‐caused quenching character of conventional fluorophores enables the complex with stably tracking the delivery of pDNA into cancer cells. Therefore, the newly developed PDTB complexes may be a promising candidate vector for traceable, safe, and effective gene delivery.     

PEI基因载体靶向遮蔽体系的制备和表征

将RGD短肽接枝到聚谷氨酸(PGA)上,制备了一种靶向性的基因载体遮蔽材料PGA-RGD.通过凝胶电泳实验及体外转染实验证明得出RGD的引入增加了载体材料与细胞表面受体的特异性作用,在载体表面正电荷得到遮蔽的同时,转染效率还得到了一定程度的增加.同时,对转染了48h的三元复合物进行MTT细胞毒性测试表明,PGA遮蔽的基因载体体系(PGA/PEI/DNA)和PGA-RGD遮蔽的基因载体体系(PGA-RGD/PEI/DNA)的细胞毒性均低于PEI/DNA复合物体系.本文开发的基因载体改性方法不仅可以对复合物颗粒表面的正电荷进行遮蔽,从而降低复合物体系对非目标组织的非特性异作用;同时引入的RGD靶向短肽还可以提高载体的靶向性,这一改性策略对推动阳离子聚合物基因载体在体内的应用具有重要意义.     

Surface modification of poly(methyl methacrylate) for improved adsorption of wall coating polymers for microchip electrophoresis

The development of rapid and simple wall coating strategies for high-efficiency electrophoretic separation of DNA is of crucial importance for the successful implementation of miniaturized polymeric DNA analysis systems. In this report, we characterize and compare different methods for the chemical modification of poly(methyl methacrylate) (PMMA) surfaces for the application of wall coating polymers. PMMA surfaces coated with 40 mol% diethylacrylamide and 60 mol% dimethylacrylamide are compared to the PMMA surfaces first oxidized and then coated with hydroxypropylmethyl-cellulose or poly(vinyl alcohol) (PVA). PMMA oxidation was accomplished with UV/ozone or an aqueous solution of HNO(3) to yield hydrogen-bond donors for the spontaneous adsorption of the coating polymers. Contact angle measurements of UV/ozone exposed PMMA surfaces indicate increase in hydrophilicity, and polymer coated surfaces show a strong dependence on the coating polymer and the oxidation method. Fast and repeatable electrophoretic separations of a 10-base and 20-base DNA ladder were performed in PMMA micro CE devices. All analyses were completed in less than 10 min, resulting in the number of theoretical plates as high as 583 000 in a 7.7 cm long separation channel. The duration of UV/ozone treatment was found to have a considerable impact on separation performance. The microchips irradiated with UV for 10 min and coated with PVA as well as the microchips treated with HNO(3) and coated with HPMC were found to have the best separation performance. These results demonstrate facile and robust methods for the surface modification of PMMA enabling low-cost single use devices for electrophoretic DNA separations.     

Self‐Assembly Pluronic and β‐Cyclodextrin to Hollow Nanospheres for Enhanced Gene Delivery

This paper studies a kind of hollow nanospheres prepared by self‐assembly β‐cyclodextrins (β‐CDs) and poly(ethylene oxide)‐poly(propylene oxide)‐poly(ethylene oxide) (pluronic F127) for gene delivery. It was found that this kind of hollow nanospheres enable load PEI10K/DNA and the resulting F127 NH₂ βCD/(PEI10K/DNA) with 0.08 µg/well DNA display equal or higher gene delivery capability compared to PEI10K/DNA with 1 µg/well DNA in the absence or presence of serum. The cytotoxicity of the nanospheres was over 100 times lower than that of PEI10K.

     

Direct Observation of Dynamic Mechanical Regulation of DNA Condensation by Environmental Stimuli

Gene delivery is a promising way to treat hereditary diseases and cancer; however, there is little understanding of DNA:carrier complex mechanical properties, which may be critical for the protection and release of nucleic acids. We applied optical tweezers to directly measure single‐molecule mechanical properties of DNA condensed using 19‐mer poly‐L ‐lysine (PLL) or branched histidine–lysine (HK) peptides. Force–extension profiles indicate that both carriers condense DNA actively, showing force plateaus during stretching and relaxation cycles. As the environment such as carrier concentration, pH, and the presence of zinc ions changes, DNA:HK complexes showed dynamically regulated mechanical properties at multiple force levels. The fundamental knowledge from this study can be applied to design a mechanically tailored complex which may enhance transfection efficiency by controlling the stability of the complex temporally and spatially.     

Synthesis of cross‐linked homopolymers and copolymers of 1‐vinylimidazole in supercritical carbon dioxide for removal of Cr(VI) from aqueous solution

In this work, cross‐linked poly(1‐vinylimidazole) (PVIm) and poly(1‐vinylimidazole‐co‐acrylic acid) [poly(VIm‐co‐AA)] polyampholytes microgel were synthesized by precipitation polymerization in supercritical carbon dioxide at 14 and 20 MPa. The obtained products were characterized by Fourier transform infrared spectroscopy and Energy‐dispersive X‐ray spectroscopy. The results indicate that the functional monomers including 1‐VIm and AA were cross‐linked in polymers successfully. Aggregate particles with diameters of approximately 200 nm were observed by scanning electron microscopy and particle size distribution. Surface area analysis showed that the surface area of microgel prepared at a pressure of 14 MPa are 40.28 m²/g. The swelling behavior of polyampholytes microgel was studied. The cross‐linking degrees of PVIm and copolymers can reach 92.6% and 98.1%. The effect of pH values of solution, contact time, adsorbent dosage, initial Cr(VI) concentration on adsorption capacity were also investigated. The results revealed that cross‐linked polymer has a high adsorption capacity of 306.0 mg/g for Cr(VI) in 130 mg/l solution at pH 2 and cross‐linker ratio of 27.3%. Copyright © 2013 John Wiley & Sons, Ltd.     

Investigation of complex formation between poly(N-vinyl imidazole) and various metal ions using the molar ratio method

The complex formation of poly(N-vinyl imidazole) (PVIm) with various metal ions was studied. UV-vis spectroscopy was employed to study the interaction of PVIm and metal ions in aqueous solution. Formation constants of PVIm-metal complexes were calculated by applying a “mole ratio” method. The stoichiometric ratios between polymer and metal ions were found to be␣4. The stability constants for the complexes of PVIm with bivalent transition metal ions were in agreement with the Irving-William series. The biggest formation constant was found for the PVIm-Cu²⁺ complex system. Received: 4 January 1999 Accepted in revised form: 1 February 1999     

Improving Gene Delivery Efficiency of Bioreducible Poly(amidoamine)s via Grafting with Dendritic Poly(amidoamine)s

Dendritic poly(amidoamine)s (PAMAM)s were introduced into the side chains of disulfide‐containing poly(amidoamine)s via repetitive Michael addition and amidation. The bioreducible poly(amidoamine)s grafted with dendritic polyamidoamines showed high buffer capacity, low cytotoxicity and strong DNA binding ability at low N/P ratio. They were able to condense DNA into small sized polycation/DNA complexes, which degraded and released the incorporated DNA under reductive conditions. In comparison to the original disulfide‐containing poly(amidoamine) with aminoethyl side chain, the grafting of the bioreducible poly(amidoamine) with dendrimer greatly improved the transfection efficiencies of 293T and HeLa cells with foreign DNA at various N/P ratios. The structure–gene delivery property relations of dendrimer‐grafted polycations will provide valuable insight into the design of highly efficient and less toxic polycationic gene carriers.

     

Bioreducible cationic random copolymer for gene delivery

Cationic polymers have been widely investigated for gene delivery, although their low transfection efficiency and high cytotoxicity limit their application. We synthesized a bioreducible cationic random copolymer, poly(cystamine bisacylamide‐aminoethyl piperazine)‐co‐poly(cystamine bisacylamide‐histamine) (denoted as CBA‐AEP‐His) from N,N′‐cystamine bis acrylamide (CBA) with aminoethyl piperazine (AEP) and histamine (His). CBA‐AEP‐His copolymer possesses disulfide linkages that endow it with redox‐responsivity to the intracellular environment. This polymer efficiently condenses pZNF580 into complexes with the size of 160 ± 4 nm to 280 ± 5 nm and positive zeta potential of 20 ± 0.3 mV to 30 ± 0.4 mV. The gel‐retardation assay shows that CBA‐AEP‐His can retard pZNF580 even at a low mass ratio of 1/1. The gene complexes were triggered to release pZNF580 when exposed to the reducing environment of dithiothreitol (DTT). CBA‐AEP‐His random copolymer presented higher buffer capacity owing to its His moieties, which protected pZNF580 from DNase degradation. The gene transfection results reveal that CBA‐AEP‐His can efficiently deliver pZNF580 and transfect EA. Hy926 cells. The MTT assay indicates that CBA‐AEP‐His and its complexes exhibit lower cytotoxicity than PEI25KDa. These results illustrate that CBA‐AEP‐His had promising properties for gene delivery, which may provide a suitable platform for the development of a non‐viral gene carrier.     

Water soluble poly(histamine acrylamide) with superior buffer capacity mediates efficient and nontoxic in vitro gene transfection

Water‐soluble cationic polymers, poly(histamine acrylamide)s (PHAs), with superior buffer capacity at the endosomal pH range were designed, prepared, and investigated for non‐viral gene transfection. PHAs were obtained with molecular weights ranging from 9.2 to 28.7 kDa through controlled radical polymerization of histamine acrylamide (HA). Acid–base titration results displayed that all PHA polymers had a remarkably high buffer capacity of about 70% at pH 5.1–7.2. 12.7–28.7 kDa PHAs were able to effectively condense DNA into nano‐sized (<220 nm) polyplexes with moderate positive surface charges (+13–+19 mV) at N/P ratios ≥10/1. CCK assays indicated that polyplexes of 12.7 and 17.5 kDa PHAs were non‐toxic to COS‐7 cells up to a tested N/P ratio of 20/1. Interestingly, the in vitro transfection using pCMV‐Luc and pEGFP‐C1 plasmid DNA as reporter genes showed that polyplexes of 12.7 kDa PHA formed at an N/P ratio of 20/1 mediated efficient transfection in COS‐7 cells under 10% serum conditions, with transfection efficiencies comparable to that of 25 kDa polyethylenimine control. Their versatile design of structures, controlled synthesis, low cytotoxicity, and high transfection activity render PHA‐based cationic polymers particularly interesting for the development of safe and efficient non‐viral gene delivery systems. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011.     

Preparation and utilization of poly(methacryloylsilatrane) as a salt‐dissociation enhancer in PEO‐based polymer electrolytes

Polymers containing silatrane units were prepared by the free radical polymerization of methacryloylsilatrane (MPS), and their conductivities were evaluated. We confirmed that MPS can be polymerized without excessive decomposition of the silatrane units by the radical polymerization initiated by azobisisobutyronitrile. The chemical structure of the polymerized MPS (pMPS) was characterized by Fourier transform infrared spectroscopy, nuclear magnetic resonance spectroscopy, gel permeation chromatography, and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectroscopy. The pMPS formed a homogeneous complex with lithium trifluoromethyl sulfonate (LiOTf), although the obtained pMPS/LiOTf complex did not show conductivity. The negligible conductivity was caused by the high glass transition temperature (T_g) of the pMPS matrix, which exceeded 70°C. The pMPS was subsequently utilized as a salt‐dissociation enhancer for the poly(ethylene oxide)‐based polymer electrolyte. MPS was copolymerized with poly[methacryloyl oligo(ethylene oxide)] (pMEO) by free radical polymerization. When the pMEO incorporated a small amount of MPS units (i.e. lower than 15 mol%), the elevation in T_g was not observed, and the conductivity markedly improved. Among the series of copolymers and when compared with pristine pMEO, the copolymer containing 6.3% of MPS units had the maximum conductivity (3.1 × 10^?4 S cm^?1 at 80°C). The Vogel–Fulcher–Tammann fitting parameters showed that the conductivity was improved by the increase in the number of carrier ions. The enhancement in salt dissociation was presumably due to the homogeneous incorporation of polar MPS units. However, when the MPS unit content exceeded 15 mol%, the conductivity was lowered because of the increase in T_g. Copyright © 2013 John Wiley & Sons, Ltd.     

A new specific metal ion chelated‐poly(N‐vinylimidazole) gel sorbents for albumin adsorption‐desorption

Poly(N‐vinylimidazole) (PVIm) hydrogels were prepared by γ‐irradiating binary mixtures of N‐vinylimidazole‐water in a ⁶⁰Co‐γ source having 4.5 kGy/h dose rate. These affinity gels having different swelling ratio of Cu(II)‐chelated, Co(II)‐chelated and plain PVIm in acetate buffer were used in the albumin adsorption studies. Bovine serum albumin (BSA) adsorption on these gels from aqueous solutions containing different amounts of BSA at different pH adjusted with acetate and phosphate buffer was investigated in batch reactors. The adsorption capacities of BSA on/in the gels were decreased dramatically by increasing the ionic strength (I) adjusting with NaCl. BSA adsorption capacities of the metal ion‐chelated gels were higher than the plain PVIm gel even if the swelling ratio of the metal ion‐chelated gels was very low comparing to the PVIm gel. The rigidity of the metal ion‐chelated gel is very high and it can be used for the column applications. More than 95% of BSA were desorbed in 3 h in the desorption medium containing KSCN for PVIm gel and EDTA for metal ion‐chelated gels. These results indicate that PVIm and metal ion‐chelated PVIm gels are very efficient to remove BSA and the different metal ion‐chelated PVIm gels show different affinity for BSA or biomolecules.     

Fluorescence and visual detection of single nucleotide polymorphism using cationic conjugated polyelectrolyte

We report a simple assay for visual detection of single nucleotide polymorphisms (SNPs) with good sensitivity and selectivity. The selectivity is determined by Escherichia coli (E. coli) DNA ligase mediated circular formation upon recognition of the point mutation on DNA targets. Rolling cycle amplification (RCA) of the perfect-matched DNA target is then initiated using the in situ formed circular template in the presence of Phi29 enzyme. Due to amplification of the DNA target, the RCA product has a tandem-repeated sequence, which is significantly longer than that for the SNP strand. Direct addition of a cationic conjugated polymer of poly[9,9'-bis(6'-(N,N,N-trimethylammonium)hexyl)fluorene-co-9,9'-bis(2-(2-(2-(N,N,N-trimethylammonium)ethoxyl)-ethoxy)-ethyl)fluorene tetrabromide] containing 20 mol% 2,1,3-benzothiadiazole (PFBT(20)) into the RCA solution leads to blue-whitish fluorescent color for SNP strand and yellowish fluorescent color for amplified DNA, due to PFBT(20)/DNA complexation induced intrachain/interchain energy transfer. To further improve the contrast for visual detection, FAM-labeled peptide nucleic acid (PNA) was hybridized to each amplified sequence, which is followed by the addition of poly{2,7-[9,9-bis(6'-N,N,N-trimethylammoniumhexyl)]fluorene-co-2,5-difluoro-1,4-phenylene dibromide} (PFP). The PNA/DNA hybridization brings PFP and FAM-PNA into close proximity for energy transfer, and the solution fluorescent color appears green in the presence of target DNA with a detection limit of 1 nM, which is significantly improved as compared to that for most reported visual SNP assay.     

Complex formation and adsorption of V3+, Cr3+ and Fe3+ ions with poly(N-vinylimidazole)

The complex formation of soluble poly (N-vinylimidazole) (PVIm) with trivalent metal ions in aqueous solution was studied by using UV–vis spectroscopy. Formation constants of PVIm–metal complexes were calculated by applying the “molar ratio” method. It was found that the interaction between PVIm and trivalent metal ions follows 4(base unit):1(metal ion) stoichiometry. The stability constants for the complexes of PVIm with trivalent transition-metal ions were in agreement with the Irving– William series. The biggest formation constant was found for the PVIm–Fe³⁺ complex system. The capacity of adsorption for these metal ions was investigated using cross-linked PVIm. Cross-linked PVIm hydrogels were prepared by irradiating binary mixture of N-vinylimidazole–water with a ⁶⁰Co γ-ray source having a dose rate of 4.5 kGy/h. Adsorption studies were performed at different pH and metal ion concentrations at room temperature. It was observed that the same sequence for the metal ions was verified by adsorption studies. Received: 28 May 2001 Accepted: 29 July     

Synthesis of dual‐functional poly(6‐azidohexylmethacrylate) brushes by a RAFT agent carrying carboxylic acid end groups

Novel types of dual‐functional surface‐attached polymer brushes were developed by interface‐mediated reversible addition‐fragmentation chain transfer (RAFT) polymerization of 6‐azidohexylmethacrylate using the surface‐immobilized RAFT agent and the free initiator. The interface‐mediated RAFT polymerization produced silicon substrate coated with dual‐functional (azido groups from monomer and carboxylic acid groups from RAFT agent) poly(6‐azidohexylmethacrylate) [poly (AHMA)] with a grafting density as high as 0.59 chains/nm². Dual‐functional polymer brushes can represent an attractive chemical platform to deliberately introduce other molecular units at specific sites. The azido groups of the poly(AHMA) brushes can be modified with alkyl groups via click reaction, known for their DNA hybridization, while the carboxylic acid end groups can be reacted with amine groups via amide reaction, known for their antifouling properties. © 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2015 , 53, 1696–1706     

Polyaspartamide–Polylactide Graft Copolymers with Tunable Properties for the Realization of Fluorescent Nanoparticles for Imaging

Here, the synthesis and the characterization of novel amphiphilic graft copolymers with tunable properties, useful in obtaining polymeric fluorescent nanoparticles for application in imaging, are described. These copolymers are obtained by chemical conjugation of rhodamine B (RhB) moieties, polylactic acid (PLA), and O‐(2‐aminoethyl)‐O′‐methyl poly(ethylene glycol) (PEG) on α,β‐poly(N‐2‐hydroxyethyl)‐d,l ‐aspartamide (PHEA). In particular, PHEA is first functionalized with RhB to obtain PHEA–RhB with a derivatization degree in RhB (DD_RhB) equal to 0.55 mol%. By varying the reaction conditions, different amounts of PLA are grafted on PHEA–RhB to obtain PHEA‐RhB‐PLA with DD_PLA equal to 1.9, 4.0, and 6.2 mol%. Then, PEG chains are grafted on PHEA‐RhB‐PLA derivatives to obtain PHEA‐RhB‐PLA‐PEG graft copolymers. The preparation of polymeric fluorescent nanoparticles with tunable properties and spherical shape is described by using PHEA‐RhB‐PLA‐PEG with DD in PLA and PEG equal to 4.0 and 4.9 mol%, by following easily scaling up processes, such as emulsion‐solvent evaporation and high pressure homogenization (HPH)‐solvent evaporation techniques.