Properties of Poly(B-amino Ester)s

Properties of Poly(B-amino Ester)s The poly(b-amino ester)s, a class of biodegradable cationic polymers, were firstly  prepared by Chiellini in 198340. These polymers were based on poly(amidoamine)s  developed in 1970 by Ferruti41, that contain tertiary amines in their backbones and  can be synthesized by a simple Michael addition reaction of bifunctional amines  and bisacrylamides. However, the interest over the use of poly(b-amino ester)s rised  significantly after its use as transfection reagent at Langer Lab in 200042. The development  of poly(b-amino ester)s emerged by the need to develop a cationic polymer  for gene delivery with high transfection efficiency and long-term biocompatibility  including hydrolyzable moieties easily degradable into non-toxic small molecule  byproducts. The synthesis of this polymer can easily be accomplished: without  necessity of independent preparation of specialized monomers; the use of stoichiometric  amounts of expensive coupling reagents, or amine protecti on strategies prior  to polymerization42. The main general objective of the work of mentioned research  group was to develop a polymer-based non-viral vector more efficient and less cytotoxic  than other cationic polymers used at that time for this purpose (such as,  polyethylenimine (PEI) or poly(L-lysine) (PLL)). In fact, poly(b-amino ester) approach exhibited a particularly attractive basis for  the development of new polymer-based transfection vectors for several reasons: the  polymers contain the required amines (positive charges to complex genetic material);  readily degradable linkages (by hydrolysis of ester bonds in the polymer backbones  may increase the biodegradability and biocompatibility); and multiple analogues  could be synthesized directly from compounds commercially available (easy and inexpensive  synthesis) allowing to tune polymer properties (like buffering capacity)42. Besides being used as transfection vector, PbAEs has been also applied in others  biomedical areas, such as delivery systems for drugs43;44 or proteins45;46, magnetic  resonance imaging agents47;48, or as scaffold for tissue engineering49;50. Synthesis and main physicochemical properties of poly(b-amino ester)s The poly(b-amino ester)s are easily synthesized by the conjugate addition of a primary  amine or bis(secondary amine) and a diacrylate, in a one-step reaction without  any side product that need be removed through further purification steps. It can be  prepared without solvents, catalysts, or complex protecting group strategies42;51. Depending on the ratio of monomers during the synthesis, poly(b-amino ester)s  can be tailored to have either amine- or diacrylate-terminated chains. An excess of  either diacrylate or amine monomer results in a prevalence of acrylate- or amineterminated  poly(b-amino ester)s, respectively52;53. The synthesis is performed either neat (solvent free) or in anhydrous organic  solvents to mitigate hydrolytic degradation during synthesis42;54. Normally, experiments  using solvents occur at lower temperature and over long periods of time  compared to solvent-free formulations. Table 1.3 summarizes the main reactions for  the synthesis of PbAE and the obtained properties such as molecular weight, polydispersity  index (à ), solvent solubility or yield. The most common solvents used are dimethylsulfoxide (DMSO), chloroform  (CHCl3), or dichloromethane (CH2Cl2)57. However, others solvents have also been  used, such as methanol, N,N-dimethylformamide (DMF) or N,N-dimethylacetamide  (DMA)59;61–63. The solvent used has influence on the final molecular weight of the  PbAE. For example, the use of CH2Cl2 typically yields higher molecular weight  polymer compared to THF42. On the other hand, solvent-free polymerizations maximize monomer concentrations,  thus favoring the intermolecular addition over intramolecular cyclization reaction64. The absence of solvent also allows rising temperature resulting in a higher  reaction rate and a lower viscosity of the reacting mixture, assisting to compensate  the higher viscosity found on the solvent-free systems. The combination between  increased monomer concentration and reaction temperature resulting in a reduction  in the reaction time64. The solvent-free reactions also allows the generation of higher  molecular weight polymers, besides increasing the reaction rate and obviating the  solvent removal step53;64. After polymerization, PbAE can be precipitated, normally in cold diethyl ether,  hexane42, ether65 or ethyl ether58 and/or then dried under vacuum57;65. Frequently, PbAEs are immediately used or stored in the cold conditions (4 _C52;66;67, 0 _C62, or  -20 _C68–70). Some PbAEs should be also kept airproof due to its strong moisture  absorption ability and easy degradation71. Concerning to the biodegradation and biocompatibility, PbAEs have been shown  generally to possess low cytotoxicity and good biocompatibility42;52;61;55;72. Different  studies have suggested that PbAEs are significantly less toxic than currently available  cationic polymers, such as, PEI and PLL51;64. Nevertheless, the increase of the  number of carbons in the backbone or side chain is associated to the increase of the  cytotoxicity73. PbAE degrade under physiological conditions via hydrolysis of their  backbone ester bonds to yield small molecular weight b-amino acids biologically  inert derivatives42;51;55;74. Some results revealed that the degradation rate of poly(b  amino ester)s was highly dependent on the hydrophilicity of the polymer, i.e., the  more hydrophilic the polymer is, the faster the degradation occurs75;76. In Table 1.4 are summarized the main characteristic of PbAEs which make them  a promising polymeric non-viral vector for gene delivery. Combinatorial libraries a fast and efficient way to evaluate different poly(bamino ester)s A fast and efficient way to study the relationships between structure and function  in particular material that could be prepared with different reagents is using combinatorial  libraries. Due to promising preliminar results of PbAEs as non-viral vectors,  Langer research group reported a parallel approach for the synthesis of hundreds of  PbAEs with different structures and the application of these libraries to a rapid and  high throughput identification of new transfection reagents and structure-function trends. For this purpose, major contributions have been reported52;53;57;66;67;72;75;77;78  not only exploring the possible structure/function relationships, but also imposing  an assortment of monomers (amines were denoted by numbers and acrylates by latin  alphabet letters) used in order to facilitate cataloging of different PbAEs (Table 1.5  and Tables A.1 and A.2 (Appendix A)). The first initial library screening was synthesized in 2001 by Lynn51. 140 Different  PbAEs from 7 diacrylates and 20 amines were prepared with molecular weights  between 2,000 and 50,000 g.mol-1. From this, polymers C93 (Mw = 3180 g.mol-1) and  G28 (Mw = 9170 g.mol-1) revealing transfection levels 4-8 times higher than control  experiments employing PEI. At same time, it was observed that for transfection efficiency,  high molecular weight was not an important parameter. This work was then  completed in 2003 by Akinc57, where biophysical properties and the ability of each  polymer/DNA complex to overcome important cellular barriers to gene deliver were investigated. As previous experiments, complexes formed from polymers C93 and  G28, revealed higher levels of internalization compared to †naked† DNA, displaying  18- and 32-fold more internalization, respectively. In contrast, the majority of the  polyplexes were found to be uptake-limited. Regarding d iameter and zeta potential,  out of 10 polymer/DNA complexes with the highest internalization rates, all  had diameters lower than 250 nm and 9 had positive zeta potentials. By measuring  the pH environment of delivered DNA through fluorescence-based flow cytometry  protocol using plasmid DNA covalently labeled with fluorescein (pH sensitive) and  Cy5 (pH insensitive) it was possible to investigate the lysosomal trafficking of the  polyplexes. The results demonstrated that complexes based on polymers C93 and  G28 were found to have near neutral pH measurements, indicating that they were  able to avoid acidic lysosomal trafficking. In the same year, Akinc64 studied the  effect of polymer molecular weight, polymer chain end-group, and polymer/DNA  ratios on in vitro gene delivery. For this purpose, 12 different structures were synthesized  based only in two different PbAE (C28 prepared from 1,4-butanediol diacrylate  and 1-aminobutanol and E28 prepared from 1,6 -hexanediol diacrylate and  1-aminobutanol) (Figure 1.6.) These structures were synthesized by varying amine/diacrylate stoichiometric ratios, resulting in PbAEs with either acrylate or amine end-groups and with molecular  weights ranging from 3,350 to 18,000 g.mol-1. Polymers were then tested, using high  throughput methods, at nine different polymer/DNA ratios between 10/1 (w/w)  and 150/1 (w/w). Concerning terminal groups, it was found that amino-terminated  polymers transfected cells more effectively than acrylate-terminated polymers. In  contrast, none of the acrylate terminated PbAEs mediated appreciable levels of  transfection activity under any of the assessed conditions. These findings suggest that end-chains of PbAE have crucial importance in transfection activity. Concerning  molecular weight effect, highest levels of transfection occurred using the higher  molecular weight samples of both amine-terminated C28 (Mw _13100 g.mol-1 and  E28 (Mw _13400 g.mol-1). Regarding the optimal polymer/DNA ratios for these   polymers, it was observed a markedly difference, 150/1 (w/w) for C28 and 30/1 for  E28. These results highlighted the importance of polymer molecular weight, polymer/DNA ratio, and the chain end-groups in gene transfection activity. Moreover, it  has found the fact that two similar polymer structures, differing only by two carbons  in the repeating unit, have different optimal transfection parameters emphasizing  the usefulness of library screening to perform these optimizations for each unique  polymer structure. Meanwhile, in 2003, Anderson52 described, for the first time,  a high-throughput and semi-automated methodology using fluid-handling systems  for the synthesis and screening of a library of PbAEs to be used as gene carrier. A crucial feature of these methods was that all process of synthesis, storage, and  cell-based assays were performed without removing solvent (DMSO). By using these  methods, it was possible to synthesize a library of 2350 structurally unique, degradable  and cationic polymers in a single day and then test those as transfection reagent  at a rate of _1000 per day. Among PbAEs tested, it was identified 46 polymers  that transfect in COS-7 as good as or better than PEI. The common characteristic  among them was the use of a hydrophobic diacrylate monomer. Moreover, in the  hit structures mono- or dialcohol side groups and linear, bis(secondary amines) are  over represented. From data obtained from this library, Anderson67, in 2004, continued  his study developing a new polymer library of >500 PbAE using monomers  that led higher transfection efficiency in the previous studies and optimizing their  polymerization conditions. The top performing polyplexes were asses sed by using  an in vitro high-throughput transfection efficiency and cytotoxicity assays at different N/P ratios. As previously observed, the most promising polymers are based on  hydrophobic acrylates and amines with alcohol groups. Among those, C32 stood  out due to higher transfection activity with no associated cytotoxicity. The efficiency  to deliver DNA was evaluated in mice after intra-tumoral (i.t.) and intra-muscular  (i.m.) injection. The results revealed important differences. While by i.t injection  C32 delivered DNA 4-fold better than jetPEI R , a commercial polymeric non-viral  vector, by i.m. administration transfection was rarely observed. C32 was then assessed  for DNA construct encoding the DT-A (DT-A DNA) deliver to cells in culture  and to xenografts derived from androgen-sensitive human prostate adenocarcinoma  cells (LNCaP). Results showed that DT-A DNA was successfully delivered and the  protein expressed in tumor cells in culture. In hu man xenografts, the growth was  suppressed in 40% of treated tumors. The fact of C32 is non-toxic and it is able to  transfect efficiently tumors locally and transfects healthy muscle poorly turned it as  a promising carrier for the local treatment of cancer. From here, a panoply of results based in PbAE combinatorial library appeared. In  2005, Anderson53, prepared a new library of 486 second-generation PbAE based on  polymers with 70 different primary structures and with different molecular weights. These 70 polymers were synthesized using monomers previously identified as common  to effective gene delivery polymers. This library was then characterized by  molecular weight of polymers, particle size, surface charge, optimal polymer/DNA  ratio and transfection efficiency in COS-7 of polymer/DNA complexes. Results  showed that from 70 polymers with primary structures, 20 possess transfection activities  as good as or better than Lipofectamine R 2000, one of the most effective commercially  available lipid reagents. Results also revealed that, in general, the most  effective polymers/DNA complexes had In 2006, Green79, synthesized, on a larger scale and at a range of molecular  weights, the top 486 of 2350 PbAEs previously assessed52 and studied their ability to  deliver DNA. These PbAEs were tested, firstly, on the basis of transfection efficacy in  COS-7 cells in serum-free conditions, and then, the 11 of the best-performing PbAEs  structures were further analyzed. The transfection conditions were optimized in human  umbilical vein endothelial cells (HUVECs) in the presence of serum. In this  study, the influence of the factors like polymer structure and molecular weight, and  biophysical properties of the polyplexes (such as, particle size, zeta potential, and  particle stability throughout time) were studied. The results showed that many of  the polyplexes formed have identical biophysical properties in the presence of buffer,  but, when in the presence of serum proteins their biophysical properties changed differentially,  influencing the transfection ac tivity. Concerning to the size, the results  showed that in spite of all vectors condensed DNA into small particles below 150 nm  in buffer, only a few, such as C32, JJ32 and E28, formed small (_200 nm) and stable  particles in serum. C32, JJ32 and E28 revealed also high transfection activity both  in the absence of serum in COS-7 cell line as in the presence of serum in HUVEC  cell line. Moreover, C32 transfected HUVECs in the presence of serum significantly  higher than jetPEI R and Lipofectamine R 2000, the two top commercially available  transfection reagents. The 3 mentioned PbAEs share a nearly identical structure. The acrylate monomers of these polymers, C, JJ, and E, differ by only their carbon  chain lengths (4, 5, and 6 carbons, respectively). Similarly, amines 20, 28, and 32  differ also by only the length of their carbon chain (3, 4, and 5 carbons, respectively). For example, polymers prepared with the same acrylate monomer (C) in which itwas increased the length of the carbons chain of the amine monomer resulted in  an increased transfection efficacy (C32 (5 carbons) > C28 (4 carbons) > C20 (3 carbons))  of these polymers-based polyplexes. Interestingly, this study reinforced C32  as the lead PbAE vector and revealed other potential two, JJ28 and E28, which previously  showedto be poor vectors. On the other hand, C28 and U28, previously  recognized as an efficient transfection reagent, were found to transfect inefficiently  HUVEC in serum. By constructing a new library of end-modified PbAE, the research  was continued78 in order to understand the structure-function relationship  of terminal modification of PbAE in transfection activity. For this purpose, it was  used twelve different amine capping reagents to end-modify C32, D60 and C20. The  choice of these 3 PbAEs was based in their transfection activity: C32, the most effective; D60, an effective transfection reagent with a significantly different structure  from that of C32; and, C20, a poor transfection reagent but with similar structure  to C32 differing only in the length of the amine monomer. The results showed  that some PbAEs-based vectors (C32-103 and C32-117) were able to deliver DNA by  approximately two orders of magnitude higher than unmodified C32, PEI (25,000  g.mol-1) or Lipofectamine R2000, and, at levels comparable to adenovirus at a reasonably  high level of infectivity (multiplicity of infection = 100). Once again, it was  demonstrated that small structural changes influence greatly gene delivery, from biophysical  properties (such as, DNA binding affinity, particle size, intracellular DNA  uptake) until final protein expression. From these 3 polymers assessed, C20 was the  one who transfected cells much less effectively, although it has seen a remarkably  improvement with end-modifications. As expected, C 32-based polyplexes, based on  C32-103 and C32-117, revealed the higher transfection efficiency enhancing cellular  DNA uptake up to five-fold compared to unmodified C32. Interestingly, and in a  general way, terminal modifications of C32 with primary alkyl diamines were more  effective than those with PEG spacers, revealing that a degree of hydrophobicity at  the chain ends is an added value for these polymers. Another interesting fact in terminal  modification of C32 was that at least a three carbon spacer between terminal  amines is necessary to obtain an efficient gene delivery. For example, results showed  that C32-103 transfection efficiency is 130- and 300-fold higher than C32-102 on the  COS-7 and HepG2 cell lines, respectively. As the molecular weight was the same,  this result demonstrated the critical role of the chain ends in transfection activity. In order to better understand the role of the chain ends in transfection efficiency  a new library of end-modified C32 was synthesized by Zugates80 in 2007 using 37  different amine molecules to end-modify the PbAE. In a general way, it was observed  that polymers end-capped with hydrophilic amine end groups containing  hydroxyls or additional amines led to higher transfection efficiency. On the other  hand, terminal-modifications with hydrophobic amines containing alkyl chains or  aromatic rings proved to be much less effective. Concerning to cytotoxicity, terminal  modification with primary monoamine reagents (independently of functional group  extending from the amine, such as aromatic, alkyl, hydroxyl, secondary and tertiary