Synthesis and Self-Assembly of β-Cyclodextrin Terminated DMA/NIPAM Diblock Copolymers

A series of β-cyclodextrin (β-CD) terminated diblock copolymers has been prepared via click reaction. The Huisgen cycloaddition between alkyne decorated copolymer and azide functionalized β-CD was performed in organic solvent in the presence of a Cu(I) catalyst, resulting in the formation of β-CD terminated diblock copolymers, which contain thermally responsive poly(N-isopropylacrylamide) (PNIPAM) block and hydrophilic poly(N,N-dimethylacrylamide) (PDMA) block. Using dynamic light scattering and fluorescence spectroscopy measurements, it is demonstrated that these β-CD functionalized block copolymers are capable of reversibly forming micelles in response to changes in solution temperature and that the critical micelle concentration, micellar size, and transition temperature are dependent on both the NIPAM block length and the polymer functionalization.     

Poly[N-(2-hydroxypropyl)methacrylamide-block-n-butyl acrylate] micelles in water/DMF mixed solvents

Three diblock copolymers of poly[N-(2-hydroxypropyl)methacrylamide] (poly(HPMA)) and poly(n-butyl acrylate) (poly(BA)) with varying lengths of blocks were prepared by atom transfer radical polymerization. All copolymers were found to be soluble in dimethylformamide (DMF) and poorly soluble or insoluble in water. In water and mixed DMF/H₂O solvents, the copolymers were dispersed in micellar form by controlled addition of water to DMF solutions of copolymers under continuous intensive stirring. The micellar solutions in water were prepared by dialysis of solutions in DMF/H₂O (95 vol% of H₂O) against water. Solution properties of diblock copolymers of poly(HPMA) and poly(BA) were studied using static and dynamic laser light scattering to characterize the behavior of the copolymers at the supramolecular level. The effects of preparation mode, organic solvent (DMF) and copolymer chemical composition on the formation of micelles were studied. While a slower mixing procedure was optimal for copolymers with short poly(HPMA) blocks, a faster mixing was more suitable for copolymers having longer poly(HPMA) blocks. Finally, the dimensions of micelles in water were evaluated. The most compact micelles were prepared from copolymers having short hydrophilic poly(HPMA) blocks. On the other hand, the copolymer with the longest poly(HPMA) block formed micelles with the smallest size and the lowest density.     

Structural effect of a series of block copolymers consisting of poly(N-isopropylacrylamide) and poly(N-hydroxyethylacrylamide) on thermoresponsive behavior

Well-defined diblock and triblock copolymers consisting of poly(N-isopropylacrylamide) (PNIPAM) and poly(N-hydroxyethylacrylamide) (PHEAA) were prepared using the atom transfer radical polymerization (ATRP) method. The number-average molecular weight and fraction of each segment were precisely controllable by adjusting the monomer/initiator ratio in feed. The lower critical solution temperature (LCST) of a series of block copolymers with different compositions was examined using a turbidimetry analysis. The copolymers with a relatively lower molar fraction of HEAA units in the polymer chain exhibited phase transition phenomenon, in which the LCST depended on the fraction in the copolymer. On the other hand, the LCST disappeared for the copolymers with higher HEAA unit molar fractions. The ¹H NMR measurement clarified that the disappearance of the LCST was attributed to the formation of the water-soluble micelle. Furthermore, the thermoresponsive property of the series of block copolymers was elucidated on the basis of the structural effect of the copolymer, which includes the order and length of the block segments.     

Novel triblock copolymers synthesized via radical telomerization of N-isopropylacrylamide in the presence of polypseudorotaxanes made from thiolated PEG and α-CDs

A protocol for the preparation of novel triblock copolymers comprising a polyrotaxane center block and outer blocks of poly(N-isopropylacrylamide) (PNIPAAm) as bulky stoppers was developed, in which N-isopropylacrylamide was allowed to telomerize in the presence of polypseudorotaxanes made from the self-assembly of thiol end-capped PEG with a varying amount of α-CDs under UV irradiation in aqueous solution. The molecular structure of the resulting copolymers was characterized in detail by ¹H NMR, FTIR, XRD, TG and DSC analyses. It was demonstrated that the PNIPAAm blocks are successfully attached to the two terminals of the polypseudorotaxanes and each block having the minimum 7 NIPAAm units seems long and bulky enough to efficiently impede the dethreading of α-CDs from the PEG axle to give rise to the triblock polyrotaxane-containing copolymers.     

DNA-functionalized thermoresponsive bioconjugates synthesized via ATRP and click chemistry

Diblock and miktoarm star-shaped thermoresponsive copolymers composed of single-stranded DNA (ssDNA) and poly(N-isopropylacrylamide) were successfully synthesized with combination of atom transfer radical polymerization (ATRP) and click chemistry. This approach should be generalizable to other DNA-functionalized copolymers. Such copolymers self-assemble into spherical micelles with ssDNA corona in aqueous solution above the lower critical solution temperature. The micellar size can be tuned from the macromolecular architecture. These DNA-encoded micellar particles are able to encapsulate and release hydrophobic guest molecules upon changing temperature.     

Doubly thermo-responsive brush-linear diblock copolymers and formation of core-shell-corona micelles

Doubly thermo-responsive brush-linear diblock copolymer of poly[poly(ethylene glycol) methyl ether vinylphenyl]-block-poly(N-isopropylacrylamide) (PmPEGV-b-PNIPAM) is prepared by RAFT polymerization. The obtained brush-linear diblock copolymer exhibits two lower critical solution temperatures (LCSTs) corresponding to the linear poly(N-isopropylacrylamide) (PNIPAM) block and the brush poly[poly(ethylene glycol) methyl ether vinylphenyl] (PmPEGV) block in water. This brush-linear diblock copolymer undergoes a two-step temperature sensitive micellization. At temperature above the first LCST, the brush-linear diblock copolymer self-assembles into core-corona micelles with the dehydrated PNIPAM block forming the core and the solvated brush PmPEGV block forming the corona. When temperature increases above the second LCST, the polystyrene backbone in the brush PmPEGV block collapses onto the dehydrated PNIPAM core to form core-shell-corona micelles, in which the dehydrated PNIPAM block forms the core, the collapsed polystyrene backbone in the brush PmPEGV block forms the shell and the solvated poly(ethylene glycol) side-chains forms the corona. The effect of the length of the PNIPAM block and the length of the poly(ethylene glycol) side-chains on the thermo-responsive micellization and the size of core-shell-corona micelles is investigated.     

Micellization and phase transition behavior of thermosensitive poly(N-isopropylacrylamide)-poly(?-caprolactone)-poly(N-isopropylacrylamide) triblock copolymers

Thermosensitive triblock copolymers with two hydrophilic poly(N-isopropylacrylamide) blocks flanking a central hydrophobic poly(?-caprolactone) block were synthesized by atom transfer radical polymerization. Core-shell micellization of the triblock copolymers was inferred from the ¹H NMR spectra derived in two different solvent environments (CDCl₃ and D₂O). The micellar characteristics of these amphiphilic triblock copolymers were studied by pyrene fluorescence techniques, dynamic light scattering and transmission electron microscopy. The critical micelle concentrations of the triblock copolymers were in the range of 4-16 mg/L and the partition coefficients were in the range of 3.10 × 10⁴ to 2.46 × 10⁵. The mean diameters of the micelles, measured by light scattering, were between 90 and 120 nm. The temperature sensitivity of the triblock copolymers was demonstrated by the phase transition of a 250 mg/L aqueous polymer solution at the lower critical solution temperature (LCST). The enthalpy of the phase transition was determined by differential scanning calorimetry. PM3 quantum mechanical calculation method was used to understand the intermolecular interactions between the copolymer and the water molecules. A modular approach was used to simulate the phase transition observed at the LCST.     

Vesicular and micellar self‐assembly of stimuli‐responsive poly(N‐isopropyl acrylamide‐b‐9‐anthracene methyl methacrylate) amphiphilic diblock copolymers

Dually responsive amphiphilic diblock copolymers consisting of hydrophilic poly(N‐isopropyl acrylamide) [poly(NIPAAm)] and hydrophobic poly(9‐anthracene methyl methacrylate) were synthesized by reversible addition fragmentation chain‐transfer (RAFT) polymerization with 3‐(benzyl sulfanyl thiocarbonyl sulfanyl) propionic acid as a chain‐transfer agent. In the first step, the poly(NIPAAm) chain was grown to make a macro‐RAFT agent, and in the second step, the chain was extended by hydrophobic 9‐anthryl methyl methacrylate to yield amphiphilic poly(N‐isopropyl acrylamide‐b‐9‐anthracene methyl methacrylate) block copolymers. The formation of copolymers with three different hydrophobic block lengths and a fixed hydrophilic block was confirmed from their molecular weights. The self‐assembly of these copolymers was studied through the determination of the lower critical solution temperature and critical micelle concentration of the copolymers in aqueous solution. The self‐assembled block copolymers displayed vesicular morphology in the case of the small hydrophobic chain, but the morphology gradually turned into a micellar type when the hydrophobic chain length was increased. The variations in the length and chemical composition of the blocks allowed the tuning of the block copolymer responsiveness toward both the pH and temperature. The resulting self‐assembled structures underwent thermally induced and pH‐induced morphological transitions from vesicles to micelles and vice versa in aqueous solution. These dually responsive amphiphilic diblock copolymers have potential applications in the encapsulation of both hydrophobic and hydrophilic drug molecules, as evidenced from the dye encapsulation studies. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018 , 135, 46474.     

Synthesis of thermoresponsive poly(N-isopropylmethacrylamide) and poly(acrylic acid) block copolymers via post-functionalization of poly(N-methacryloxysuccinimide)

Polymers exhibiting a thermoresponsive, lower critical solution temperature (LCST) phase transition have proven to be useful for many applications as “smart” or “intelligent” materials. A series of poly(N-isopropylmethacrylamide) (PNIPMAM) polymer, poly(N-isopropylmethacrylamide)-b-poly(acrylic acid) (PNIPMAM-b-PAA) diblock, and poly(acrylic acid)-b-poly(N-isopropylmethacrylamide)-b-poly(acrylic acid) (PAA-b-PNIPMAM-b-AA) triblock copolymer samples were synthesized via ATRP. A facile post-functionalization route was developed that uses an activated ester functionality to convert poly(N-methacryloxysuccinimide) (PMASI) blocks to LCST capable polyacrylamide, while poly(t-butyl acrylate) (PtBA) blocks were converted to water-soluble poly(acrylic acid) (PAA). The post-functionalization was monitored via ¹H NMR and ATR-FTIR. The aqueous solution properties were explored and the PNIPMAM polymers were shown to have a LCST phase transition varying from 35 to 60 °C. The ability to synthesize block copolymers that are thermoresponsive and water-soluble will be of great benefit for broader applications in drug delivery, bioengineering, and nanotechnology.     

pH或/和温度双重响应嵌段接枝膜的开关特性

利用原子转移自由基聚合法(ATRP)成功地制备了聚甲基丙烯酸-嵌段-聚N-异丙基丙烯酰胺(PMN)和聚N-异丙基丙烯酰胺-嵌段-聚甲基丙烯酸(PNM)接枝开关膜。通过通量实验系统考察了两类开关膜分别或同时对pH和温度的响应性。结果表明:用ATRP法接枝嵌段共聚物开关中第一段接枝物的接枝率总是高于第二段接枝物的接枝率;该嵌段接枝开关膜对pH和温度同时响应的开关系数要大于其对单一pH或温度响应的开关系数;嵌段接枝开关中第一段接枝物对膜孔的"开"或"关"起主导作用,而第二段接枝物的影响相对较小。实验结果还表明,PMAA的pH响应开关系数比PNIPAM的温度响应开关系数显著。研究结果为设计和制备双重或多重嵌段接枝开关膜提供了有价值的参考。     

Synthesis, characterization and self-assembly behavior in water as fluorescent sensors of cationic water-soluble conjugated polyfluorene-b-poly(N-isopropylacrylamide) diblock copolymers

A series of well-defined novel water-soluble diblock copolymers containing conjugated amino-terminal polyfluorene (PF) block and coil-like poly(N-isopropylacrylamide) (PNIPAM) have been successfully synthesized through atom transfer radical polymerization (ATRP) initiated by a 2-bromoisobutyrate end-capped PF macroinitiator using CuCl/HMTETA (1,1,4,7,10,10-hexamethyltriethylenetetramine) as the catalyst. The first-order kinetic plots indicate the presence of a constant number of active species during the polymerization. The molecular weight and molecular weight distribution can be well controlled, implying synthesis of well-defined block structures of the copolymers. The chemical structures of block copolymers have been characterized by ¹H NMR, UV-vis, and photoluminescence (PL) spectra. PF aggregates are formed in water with the increase of temperature for the conjugated-ionic diblock copolymer synthesized, as confirmed by dynamic light scattering (DLS). The formation of excimers within the PF aggregates results in improved Förster resonance energy transfer (FRET) efficiencies.     

Effect of block compositions of amphiphilic block copolymers on the physicochemical properties of polymeric micelles

Amphiphilic block copolymers with various chain lengths of poly(n-butyl methacrylate) blocks (PBMA) and poly(N-acryloylmorpholine) blocks (PAM) were prepared by RAFT polymerization. Packing parameter of block polymers in water (β < 0.2) indicated the formation of core-corona structures, which was further confirmed from a difference between core- and corona-forming chain surface areas. Hydrodynamic micellar size was related with the numbers of BMA (N_BMA) and AM (N_AM), and their ratios (N_BMA/N_AM). With increasing N_BMA/N_AM value, the polymer aggregation numbers and inner core sizes increased, while the critical micelle concentrations, the corona thickness, and the second virial coefficient of block copolymer micelles decreased. These properties changed with increasing N_BMA/N_AM value resulted in a linear increase in corona chain unit density (ρ_AM) that limited chain mobility. Thus, the interaction between the micelles and serum protein at low ρ_AM disappeared at a higher value. Consequently, both micellar properties and biocompatible effect can be regulated by tailoring the block compositions of amphiphilic polymers.     

Synthesis of complex architectures of comb block copolymers

The synthesis of comb block copolymers by ring opening metathesis polymerization (ROMP), ring opening polymerization (ROP), and atom transfer radical polymerization (ATRP) is described. Block copolymers were synthesized by the ROMP of oxanorbornene and norbornene monomers followed by hydrogenation of the olefins along the backbone. One block of these diblock copolymers possessed initiators either for the ROP of (3S)-cis-3,6-dimethyl-1,4-dioxane-2,5-dione or the ATRP of butyl acrylate. The synthesis and characterization of comb polymers with arms composed of poly(lactic acid) and poly(butyl acrylate) are described. These polymers had well-defined peaks in the size exclusion chromatography spectra which indicated that no homopolymers were synthesized. A comb block copolymer with polymeric arms of poly(styrene-b-vinylpyridine) is described. Vinylpyridine was polymerized from a comb polymer with poly(styrene) arms by ATRP at high dilution of the comb polymer.     

Synthesis and characterization of amphiphilic block copolymers from poly(ethylene glycol)methyl ether and 4-methyl-?-caprolactone or 4-phenyl-?-caprolactone

MPEG-b-PMCL and MPEG-b-PBCL diblock copolymers were synthesized by ring-opening polymerization of 4-methyl-?-caprolactone (MCL) or 4-phenyl-?-caprolactone (BCL) using monomethoxy poly(ethylene glycol) (MPEG, M_n = 550 or 2000 g mol⁻¹) as the macroinitiator and SnOct₂ as the catalyst. These copolymers were characterized by differential scanning calorimetry (DSC), ¹H NMR, ¹³C NMR, and gel permeation chromatography. The thermal properties (T_g and T_m) of the diblock copolymers depend on the composition of polymers. When larger amount of MCL or BCL was incorporated into the macromolecular backbone there was an increase in T_g. Their micellar characteristics in the aqueous phase were investigated by fluorescence spectroscopy, transmission electron microscopy (TEM), and dynamic light scattering (DLS). The block copolymers formed micelles in the aqueous phase with critical micelle concentrations (CMCs) in the range of 0.5-2.9 mg L⁻¹, depending on the composition of polymers. The lengths of hydrophilic segment influence the shape of micelle. The mean hydrodynamic diameters of micelles from DLS were in the range of 70-140 nm. The drug entrapment efficiency and the drug-loading content of micelles depending on the composition of block polymers were described.     

Synthesis and characterization of well-defined hydrophilic block copolymer brushes by aqueous ATRP

Homopolymer brushes of poly(N,N-dimethylacrylamide) (PDMA), poly(methoxyethylacrylamide) (PMEA) and poly(N-isopropylacrylamide)(PNIPAM) grown on atom transfer radical polymerization (ATRP) initiator functionalized latex particles were used as macroinitiators for the synthesis of PDMA-b-PNIPAM/PMEA, PMEA-b-PDMA/PNIPAM and PNIPAM-b-PDMA block copolymer brushes by surface initiated aqueous ATRP. The grafted homopolymer and block copolymer brushes were analyzed for molecular weight, molecular weight distribution, chain grafting density, composition and hydrodynamic thickness (HT) using gel permeation chromatography-multi-angle laser light scattering, ¹H NMR, particle size analysis and atomic force microscopy (AFM) techniques. The measured graft molecular weight increased following the second ATRP reaction in all cases, indicating the second block had been added. Chain growth depended on the nature of the monomer used for block copolymerization and its concentration. Unimodal distribution of polymer chains in GPC with non-overlap of molar mass-elution volume curves implied an efficient block copolymerization. This was supported by the increase in HT measured by particle size analysis, equilibrium thickness observed by AFM and the composition of the block copolymer layer by ¹H NMR analysis, both in situ and on cleaved chains in solution. ¹H NMR analysis of the grafted latex and cleaved polymers from the surface demonstrated that accurate determination of the copolymer composition by this method is possible without detaching polymer chains from surface. Block copolymer brushes obey the same power law dependence of HT on molecular weight as homopolymer brushes in good solvent conditions. The NIPAM-containing block copolymer brushes were sensitive to changes in the environment as shown by a decrease in HT with increase in the temperature of the medium.     

Control of the lower critical solution temperature—type cononsolvency properties of poly(N-isopropylacrylamide) in water—dioxane mixtures through copolymerisation with acrylamide

The behaviour of the homopolymers poly(N-isopropylacrylamide) (PNIPAM), polyacrylamide (PAM) and random copolymers of N-isopropylacrylamide (NIPAM) with acrylamide (AM) was studied by turbidimetry and viscometry in mixtures of water with dioxane. It was found that the well-known lower critical solution temperature-type cononsolvency properties of PNIPAM in water-dioxane mixtures, observed in the water-rich region, can be effectively controlled by copolymerisation of NIPAM with AM. Thus, the cononsolvency properties of the copolymers in water-dioxane mixtures are shifted to higher temperatures and restricted within a narrower solvent composition region as the acrylamide content of the copolymers increases. A significant decrease of the reduced viscosity of the systems exhibiting phase separation properties was observed upon heating, indicative of the collapse of the (co)polymer chains as temperature approaches the corresponding cloud point temperature. Furthermore, when temperature is fixed close to the cloud point temperature, the reduced viscosity decreases with increasing the volume fraction of dioxane, φ, as far as the solvent mixtures are rich in water. On the contrary, the reduced viscosity of PNIPAM in dioxane-rich mixtures is found significantly higher, indicative of an expansion of the polymer chain, as compared to the reduced viscosity of this polymer in the two pure solvents.     

Synthesis of poly(<Emphasis Type="Italic">N</Emphasis>-isopropylacrylamide-<Emphasis Type="Italic">b</Emphasis>-<Emphasis Type="Italic">N</Emphasis>-vinylcarbazole) copolymers via RAFT polymerization and its stimuli responsive morphology in aqueous media

Here, we report the successful synthesis of series of stimuli responsive amphiphilic diblock copolymers (SRABCs) poly(N-isopropylacrylamide-b-N-vinylcarbazole) [poly(NIPAAm-b-NVK)] through reversible addition fragmentation chain transfer (RAFT) polymerization. Copolymers with fixed hydrophilic [poly(NIPAAm)] block length and variable (with three different) hydrophobic [poly(NVK)] block lengths were synthesized and the block length ratio was confirmed from their molecular weight data. The self-assembly nature of synthesized block copolymers was confirmed by determining critical micelle concentration (CMC). Self-assembled block copolymers showed rice-grain like morphology for copolymers having equivalent hydrophobic/hydrophilic chain length but in case of block copolymers having smaller and bigger hydrophobic chain length with respect to hydrophilic chain length displayed vesicular morphology. The thermo and pH responsiveness of the block copolymers was found to be influenced by variation in length and chemical composition of the blocks. Due to their thermo and pH responsiveness resulted self-assembled structures underwent morphology transitions from vesicular and rice grain like to micellar structure in aqueous medium. The probable applications of the studied stimuli responsive amphiphilic diblock copolymers can be found in the nanotechnology and biotechnology are indicated.

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Graphical abstract Synthesis, self-assembly and stimuli responsiveness of poly(NIPAAm-b-NVK) copolymers.

     

Synthesis and characterization of phenylene-thiophene all-conjugated diblock copolymers

Grignard metathesis (GRIM) polymerization for all-conjugated diblock copolymers comprising poly(2,5-dihexyloxy-1,4-phenylene) (PPP) and poly(3-hexylthiophene) (P3HT) blocks were systematically studied with LiCl as additive and 1,2-bis (diphenylphosphino) ethane nickel dichloride (Ni(dppe)Cl₂) or 1,3-bis(diphenylphosphino) propane nickel dichloride (Ni(dppp)Cl₂) as catalyst. It was found that the addition order of the monomers was crucial for the success of copolymerization. With the monomer addition in the order of phenyl and then thienyl Grignard reagents, all-conjugated PPP-b-P3HT diblock copolymers with different block ratios were successfully synthesized. In contrast, the inverted addition order only afforded a mixture containing both block copolymers and deactivated or end-capped homopolymers. Mass spectroscopic analysis indicates that the effect of the addition order of the monomers on copolymerization is attributed to the low efficiency of intramolecular Ni transfer from thiophene to phenylene units. The resulting PPP-b-P3HT diblock copolymers were characterized by differential scanning calorimetry (DSC) and atomic force microscopy (AFM). It was found that both PPP and P3HT blocks in the copolymers were crystalline, and microphase separation between them took place, as indicated by two endothermal transitions corresponding to the melting of PPP and P3HT blocks, respectively. These unique properties may render PPP-b-P3HT diblock copolymers potential applications in optoelectronics.     

Poly(N-tert-butyl acrylamide-b-N-acryloylmorpholine) amphiphilic block copolymers via RAFT polymerization: Synthesis, purification and characterization

Amphiphilic block copolymers consisting of two poly(acrylamide) derivative blocks have been synthesized via the reversible addition fragmentation chain transfer (RAFT) polymerization process with a hydrophobic block, poly(N-tert-butyl acrylamide), poly(TBAm), and a non-ionic hydrophilic one, poly(N-acryloylmorpholine), poly(NAM). Both polymerization orders, poly(TBAm-b-NAM) and poly(NAM-b-TBAm), were compared in terms of conversion and control over molecular weights (MW). Purification of the block copolymers was carried out via several methods in order to optimize their subsequent characterization. ¹H NMR analysis resulted in an accurate determination of the second block MW whereas determination of the CMC by the pendant drop method confirmed the ability of the poly(TBAm-b-NAM) block copolymers to self-assemble into micelles in aqueous phase.     

A novel well-defined amphiphilic diblock copolymer containing perfluorocyclobutyl aryl ether-based hydrophobic segment

A series of well-defined binary hydrophilic-fluorophilic diblock copolymers were synthesized by successive atom transfer radical polymerization (ATRP) of methoxylmethyl acrylate (MOMA) and 4-(4′-p-tolyloxyperfluorocyclobutoxy)benzyl methacrylate (TPFCBBMA) followed by the acidic selective hydrolysis of the hydrophobic poly(methoxymethyl acrylate) (PMOMA) segment into the hydrophilic poly(acrylic acid) (PAA) segment. ATRP of MOMA was initiated by 2-MBP at 50 °C in bulk to give two different PMOMA homopolymers with narrow molecular weight distributions (M_w/M_n ≤ 1.15). PMOMA-b-PTPFCBBMA well-defined diblock copolymers were synthesized by ATRP of TPFCBBMA at 90 °C in anisole using Br-end-functionalized PMOMA homopolymer as macroinitiator and CuBr/PMDETA as catalytic system. The final PAA-b-PTPFCBBMA amphiphilic diblock copolymers were obtained via the selective hydrolysis of PMOMA block in dilute HCl without affecting PTPFCBBMA block. The critical micelle concentrations (cmc) of PAA-b-PTPFCBBMA amphiphilic copolymers in aqueous media were determined by fluorescence spectroscopy using pyrene as probe and these diblock copolymers showed different micellar morphologies with the changing of the composition.