Properties of Poly(B-amino Ester)s

Properties of Poly(B-amino Ester)sThe poly(b-amino ester)s, a class of biodegrad equal cationic polymers, were firstlyprepared by Chiellini in 198340. These polymers were based on poly(amidoamine)s bringed in 1970 by Ferruti41, that contain tertiary amines in their vertebral columns andcan be synthesized by a simple Michael appendix reaction of bi puzzle outal aminesand bisacrylamides. However, the interest over the use of poly(b-amino ester)s risedsignificantly after its use as transfection reagent at Langer Lab in 200042. The developmentof poly(b-amino ester)s emerged by the need to develop a cationic polymerfor divisor delivery with heights transfection efficiency and long-term biocompatibilityincluding hydrolyzable moieties easily degradable into non-toxic small moleculebyproducts. The synthesis of this polymer can easily be accomplished withoutnecessity of independent preparation of specialized monomers the use of stoichiometricamounts of expensive coupling reagents, or ami ne protection strategies priorto polymerization42. The main oecumenical objective of the work of mentioned researchgroup was to develop a polymer-based non-viral vector more efficient and less cytotoxicthan other cationic polymers utilise 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 forthe development of new polymer-based transfection vectors for several reasons thepolymers contain the required amines (positive charges to Byzantine genetic material)readily degradable linkages (by hydrolysis of ester bonds in the polymer backbonesmay increase the biodegradability and biocompatibility) and multiple analoguescould be synthesized directly from compounds commercially on tap(predicate) ( palmy and inexpensivesynthesis) allowing to tune polymer properties (like buffering capacity)42.Besides being used as transfection vector, PbAEs has been as well applied in others biomedical areas, such as delivery systems for drugs4344 or proteins4546, magneticresonance imaging agents4748, or as scaffold for tissue engineering4950.Synthesis and main physicochemical properties of poly(b-amino ester)sThe poly(b-amino ester)s are easily synthesized by the conjugate addition of a oldamine or bis(secondary amine) and a diacrylate, in a one-step reaction withoutany side product that need be removed through further purification steps. It can beprepared without dissolvents, catalysts, or complex protecting group strategies4251.Depending on the ratio of monomers during the synthesis, poly(b-amino ester)scan be tailored to have all amine- or diacrylate-terminated chains. An excess ofeither diacrylate or amine monomer results in a prevalence of acrylate- or amineterminatedpoly(b-amino ester)s, respectively5253.The synthesis is per organise either neat (solvent free) or in anhydrous organicsolvents to mitigate hydrolytic debasement during synthesis4254. Normally, experiments apply solvents occur at lower temperature and over long periods of timecompared to solvent-free formulations. Table 1.3 summarizes the main reactions forthe synthesis of PbAE and the obtained properties such as molecular weight, polydispersityindex (), solvent solubility or yield.The most common solvents used are dimethylsulfoxide (DMSO), chloroform(CHCl3), or dichloromethane (CH2Cl2)57. However, others solvents have also beenused, such as methanol, N,N-dimethylformamide (DMF) or N,N-dimethylacetamide(DMA)596163. The solvent used has allure on the goal molecular weight of thePbAE. For example, the use of CH2Cl2 typically yields full(prenominal)er molecular weightpolymer 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 highreaction rate and a lower viscosity of the r eacting mixture, assisting to compensatethe higher viscosity found on the solvent-free systems. The combination betweenincreased monomer concentration and reaction temperature resulting in a drop-offin the reaction time64. The solvent-free reactions also allows the generation of highermolecular weight polymers, besides increasing the reaction rate and obviating thesolvent removal step5364. later on polymerization, PbAE can be precipitated, normally in cold diethyl ether,hexane42, ether65 or ethyl ether58 and/or then dried under vacuum5765. Frequently, PbAEs are immediately used or stored in the cold conditions (4 _C526667, 0 _C62, or-20 _C6870). Some PbAEs should be also kept airproof due to its strong moistureabsorption ability and easy degradation71.Concerning to the biodegradation and biocompatibility, PbAEs have been showngenerally to possess low cytotoxicity and tidy biocompatibility4252615572. Differentstudies have fireed that PbAEs are significantly less toxic than presen tly availablecationic polymers, such as, PEI and PLL5164. Nevertheless, the increase of thenumber of carbons in the backbone or side chain is associated to the increase of thecytotoxicity73. PbAE degrade under physiological conditions via hydrolysis of theirbackbone ester bonds to yield small molecular weight b-amino acids biologicallyinert derivatives42515574. Some results revealed that the degradation rate of poly(bamino ester)s was highly dependent on the hydrophilicity of the polymer, i.e., themore hydrophilic the polymer is, the faster the degradation occurs7576.In Table 1.4 are summarized the main characteristic of PbAEs which make thema promising polymeric non-viral vector for gene delivery.Combinatorial libraries a fast and efficient way to evaluate different poly(bamino ester)sA fast and efficient way to playing area the races between structure and functionin particular material that could be prepared with different reagents is using combinatoriallibraries. Due to promis ing preliminar results of PbAEs as non-viral vectors,Langer research group reported a parallel approach for the synthesis of hundreds ofPbAEs with different structures and the application of these libraries to a rapid andhigh throughput identification of new transfection reagents and structure-function trends. For this purpose, major contributions have been reported525357666772757778not only exploring the possible structure/function relationships, but also imposingan assortment of monomers (amines were denoted by numbers and acrylates by latinalphabet letters) used in order to facilitate cataloging of different PbAEs (Table 1.5and Tables A.1 and A.2 (Appendix A)).The first initial library screening was synthesized in 2001 by Lynn51. 140 DifferentPbAEs from 7 diacrylates and 20 amines were prepared with molecular weightsbetween 2,000 and 50,000 g.mol-1. From this, polymers C93 (Mw = 3180 g.mol-1) andG28 (Mw = 9170 g.mol-1) revealing transfection levels 4-8 times higher than controlex periments employing PEI. At same time, it was observed that for transfection efficiency,high molecular weight was not an master(prenominal) parameter. This work was thencompleted in 2003 by Akinc57, where biophysical properties and the ability of eachpolymer/ deoxyribonucleic acid complex to overcome important cellular barriers to gene deliver were investigated. As previous experiments, complexes formed from polymers C93 andG28, revealed higher levels of internalization compared to naked desoxyribonucleic acid, displaying18- and 32-fold more internalization, respectively. In contrast, the majority of thepolyplexes were found to be use of goods and services-limited. Regarding diameter and zeta potential,out of 10 polymer/DNA complexes with the highest internalization rates, allhad diameters lower than 250 nm and 9 had positive zeta potentials. By measuringthe pH environment of delivered DNA through fluorescence-based flow cytometryprotocol using plasmid DNA covalently labeled with fluorescein (pH sensitive) andCy5 (pH insensitive) it was possible to investigate the lysosomal trafficking of thepolyplexes. The results demonstrated that complexes based on polymers C93 andG28 were found to have nigh(a) neutral pH measurements, indicating that they wereable to avoid acidic lysosomal trafficking. In the same year, Akinc64 studied theeffect of polymer molecular weight, polymer chain end-group, and polymer/DNAratios on in vitro gene delivery. For this purpose, 12 different structures were synthesizedbased only in two different PbAE (C28 prepared from 1,4-butanediol diacrylateand 1-aminobutanol and E28 prepared from 1,6-hexanediol diacrylate and1-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 molecularweights ranging from 3,350 to 18,000 g.mol-1. Polymers were then tested, using highthroughput methods, at nine different polymer/DNA rat ios between 10/1 (w/w)and cl/1 (w/w). Concerning terminal groups, it was found that amino-terminatedpolymers transfected cells more legally than acrylate-terminated polymers. Incontrast, none of the acrylate terminated PbAEs mediated appreciable levels oftransfection activity under any of the assessed conditions. These findings suggest that end-chains of PbAE have life-or-death importance in transfection activity. Concerningmolecular weight effect, highest levels of transfection occurred using the highermolecular weight samples of both amine-terminated C28 (Mw _13100 g.mol-1 andE28 (Mw _13400 g.mol-1). Regarding the optimal polymer/DNA ratios for thesepolymers, it was observed a markedly difference, 150/1 (w/w) for C28 and 30/1 forE28. These results highlighted the importance of polymer molecular weight, polymer/DNA ratio, and the chain end-groups in gene transfection activity. Moreover, ithas found the fact that two uniform polymer structures, differing only by two carbonsin the repeating unit, have different optimal transfection parameters emphasizingthe usefulness of library screening to perform these optimizations for each crotchetypolymer structure. Meanwhile, in 2003, Anderson52 described, for the first time,a high-throughput and semi-automated methodology using fluid-handling systemsfor 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, andcell-based assays were performed without removing solvent (DMSO). By using thesemethods, it was possible to synthesize a library of 2350 structurally unique, degradableand cationic polymers in a unmarried day and then test those as transfection reagentat a rate of _1000 per day. Among PbAEs tested, it was identified 46 polymersthat transfect in COS-7 as good as or purify than PEI. The common characteristicamong them was the use of a hydrophobic diacrylate monomer. Moreover, in thehit structures mono- or di in ebriant side groups and linear, bis(secondary amines) areover represented. From data obtained from this library, Anderson67, in 2004, continuedhis study developing a new polymer library of 500 PbAE using monomersthat led higher transfection efficiency in the previous studies and optimizing theirpolymerization conditions. The top performing polyplexes were assessed by usingan in vitro high-throughput transfection efficiency and cytotoxicity assays at different N/P ratios. As previously observed, the most promising polymers are based onhydrophobic acrylates and amines with alcohol groups. Among those, C32 stoodout due to higher transfection activity with no associated cytotoxicity. The efficiencyto 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 injectionC32 delivered DNA 4-fold better than jetPEI R , a commercial polymeric non-viralvector, by i.m. administration transfection was rarely observed. C32 was then assessedfor DNA construct encoding the DT-A (DT-A DNA) deliver to cells in husbandryand to xenografts derived from androgen-sensitive human prostate adenocarcinomacells (LNCaP). Results showed that DT-A DNA was successfully delivered and theprotein expressed in tumor cells in culture. In human xenografts, the growth wassuppressed in 40% of treated tumors. The fact of C32 is non-toxic and it is able totransfect efficiently tumors locally and transfects healthy muscle poorly turned it asa promising carrier for the local treatment of cancer.From here, a panoply of results based in PbAE combinatorial library appeared. In2005, Anderson53, prepared a new library of 486 second-generation PbAE based onpolymers with 70 different primary structures and with different molecular weights.These 70 polymers were synthesized using monomers previously identified as commonto effective gene delivery polymers. This library was then characterized bymolecular weight of po lymers, particle size, surface charge, optimal polymer/DNAratio and transfection efficiency in COS-7 of polymer/DNA complexes. Resultsshowed that from 70 polymers with primary structures, 20 possess transfection activitiesas good as or better than Lipofectamine R 2000, one of the most effective commerciallyavailable lipid reagents. Results also revealed that, in general, the mosteffective polymers/DNA complexes had In 2006, Green79, synthesized, on a larger scale and at a range of molecularweights, the top 486 of 2350 PbAEs previously assessed52 and studied their ability todeliver DNA. These PbAEs were tested, firstly, on the basis of transfection efficacy inCOS-7 cells in serum-free conditions, and then, the 11 of the best-performing PbAEsstructures were further analyzed. The transfection conditions were optimized in humanumbilical vein endothelial cells (HUVECs) in the front man of serum. In thisstudy, the influence of the factors like polymer structure and molecular weight, andb iophysical properties of the polyplexes (such as, particle size, zeta potential, andparticle stability throughout time) were studied. The results showed that many ofthe 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 activity. Concerning to the size, the resultsshowed that in spite of all vectors condensed DNA into small particles below 150 nmin buffer, only a few, such as C32, JJ32 and E28, formed small (_200 nm) and stableparticles in serum. C32, JJ32 and E28 revealed also high transfection activity bothin the absence of serum in COS-7 cell line as in the presence of serum in HUVECcell line. Moreover, C32 transfected HUVECs in the presence of serum significantlyhigher than jetPEI R and Lipofectamine R 2000, the two top commercially availabletransfection reagents. The 3 mentioned PbAEs share a nearly identical structure.The a crylate monomers of these polymers, C, JJ, and E, differ by only their carbonchain lengths (4, 5, and 6 carbons, respectively). Similarly, amines 20, 28, and 32differ 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 inan increased transfection efficacy (C32 (5 carbons) C28 (4 carbons) C20 (3 carbons))of these polymers-based polyplexes. Interestingly, this study reinforced C32as the lead PbAE vector and revealed other potential two, JJ28 and E28, which previouslyshowedto be poor vectors. On the other hand, C28 and U28, previouslyrecognized as an efficient transfection reagent, were found to transfect inefficientlyHUVEC in serum. By constructing a new library of end-modified PbAE, the researchwas continued78 in order to understand the structure-function relationshipof terminal modification of PbAE in transfection activity. For this purpose, it wasused twelve different amine capping reagents to end-modify C32, D60 and C20. Thechoice of these 3 PbAEs was based in their transfection activity C32, the most effective D60, an effective transfection reagent with a significantly different structurefrom that of C32 and, C20, a poor transfection reagent but with similar structureto C32 differing only in the length of the amine monomer. The results showedthat some PbAEs-based vectors (C32-103 and C32-117) were able to deliver DNA byapproximately two orders of magnitude higher than unmodified C32, PEI (25,000g.mol-1) or Lipofectamine R2000, and, at levels comparable to adenovirus at a reasonablyhigh level of infectivity (multiplicity of infection = 100). Once again, it wasdemonstrated that small structural changes influence greatly gene delivery, from biophysicalproperties (such as, DNA binding affinity, particle size, intracellular DNAuptake) until final protein expression. From these 3 p olymers assessed, C20 was theone who transfected cells much less effectively, although it has seen a remarkablyimprovement with end-modifications. As expected, C32-based polyplexes, based onC32-103 and C32-117, revealed the higher transfection efficiency enhancing cellularDNA uptake up to five-fold compared to unmodified C32. Interestingly, and in ageneral way, terminal modifications of C32 with primary alkyl diamines were moreeffective than those with PEG spacers, revealing that a degree of hydrophobicity atthe chain ends is an added value for these polymers. Another interesting fact in terminalmodification of C32 was that at least a three carbon spacer between terminalamines is necessary to obtain an efficient gene delivery. For example, results showedthat C32-103 transfection efficiency is 130- and 300-fold higher than C32-102 on theCOS-7 and HepG2 cell lines, respectively. As the molecular weight was the same,this result demonstrated the critical bureau of the chain ends in tra nsfection activity.In order to better understand the role of the chain ends in transfection efficiencya new library of end-modified C32 was synthesized by Zugates80 in 2007 using 37different amine molecules to end-modify the PbAE. In a general way, it was observedthat polymers end-capped with hydrophilic amine end groups containinghydroxyls or additional amines led to higher transfection efficiency. On the otherhand, terminal-modifications with hydrophobic amines containing alkyl chains oraromatic rings proved to be much less effective. Concerning to cytotoxicity, terminalmodification with primary monoamine reagents (independently of functional groupextending from the amine, such as aromatic, alkyl, hydroxyl, secondary and tertiary