Harnessing Cytosine for Tunable Nanoparticle Self-Assembly Behavior Using Orthogonal Stimuli

Nucleobases control the assembly of DNA, RNA, etc. due to hydrogen bond complementarity. By combining these unique molecules with state-of-the-art synthetic polymers, it is possible to form nanoparticles whose self-assembly behavior could be altered under orthogonal stimuli (pH and temperature). Herein, we report the synthesis of cytosine-containing nanoparticles via aqueous reversible addition-fragmentation chain transfer polymerization-induced self-assembly. A poly(N-acryloylmorpholine) macromolecular chain transfer agent (mCTA) was chain-extended with cytosine acrylamide, and a morphological phase diagram was constructed. By exploiting the ability of cytosine to form dimers via hydrogen bonding, the self-assembly behavior of cytosine-containing polymers was altered when performed under acidic conditions. Under these conditions, stable nanoparticles could be formed at longer polymer chain lengths. Furthermore, the resulting nanoparticles displayed different morphologies compared to those at pH 7. Additionally, particle stability post-assembly could be controlled by varying pH and temperature. Finally, small-angle X-ray scattering was performed to probe their dynamic behavior under thermal cycling.


SAXS data modelling
Programming tools within the Irena SAS Igor Pro macros 1 were used to implement the scattering models.
In general, the intensity of X-rays scattered by a dispersion of nano-objects [as represented by the scattering cross-section per unit sample volume, Σ Ω (q)] can be expressed as: where (,  1 , … ,   ) is the form factor,  1, … ,   is a set of k parameters describing the structural morphology, Ψ( 1, … ,   ) is the distribution function, S(q) is the structure factor and N is the number density of nano-objects per unit volume expressed as: where ( 1 , … ,   ) is the volume of the nano-object and  is its volume fraction within the dispersion.It is assumed that S(q) = 1 in this study.

Spherical micelle model
The spherical micelle form factor for Equation S1 is given by 2 : () =   2   2   2 (,   ) +     2   (,   ) +   (  − 1)  2   2 () where   is the volume-average sphere core radius and   is the radius of gyration of the coronal steric stabilizer block (in this case, PHEMA 30 ).The X-ray scattering length contrasts for the core and corona blocks are given by   =   (  −   ) and   =   (  −   ) respectively.Here,   ,   and   are the X-ray scattering length densities of the core block ( PCAm = 10.63 x 10 10 cm -2 ), corona block ( PNAM = 10.60 x 10 10 cm -2 ) and pH2 water solvent (  = 9.42 x 10 10 cm -2 ), respectively.  and   are the volumes of the core block ( PCAm ) and the corona block ( PNAM ), respectively.The sphere form factor amplitude is used for the amplitude of the core self-term: where Φ(  ) = .A sigmoidal interface between the two blocks was assumed for the spherical micelle form factor (Equation S3).This is described by the exponent term with a width  accounting for a decaying scattering length density at the micellar interface.This  value was fixed at 2.5 during fitting.
The form factor amplitude of the spherical micelle corona is: The radial profile,   (), can be expressed by a linear combination of two cubic bsplines, with two fitting parameters  and  corresponding to the width of the profile and the weight coefficient respectively.This information can be found elsewhere, 3,4 as can the approximate integrated form of Equation S5.The self-correlation term for the coronal block is given by the Debye function: where   is the radius of gyration of the PNAM coronal block.In all cases   was fixed to be 1.6 nm, which is estimated by assuming the total contour length of PNAM 40 is 10.21 nm (40 × 0.255 nm, where 0.225 nm is the contour length of one NAM monomer unit with two C-C bonds in all-trans conformation).Given a mean Kuhn length of 1.53 nm, based on the known literature value for poly(methyl methacrylate) 5 , an estimated unperturbed   of 1.6 nm is determined using   = (10.21× 1.53/6) 0.5 .
The aggregation number,  s , of the spherical micelle is given by: where   is the volume fraction of solvent within the PCAm micelle cores, which was found to be zero in all cases.A polydispersity for one parameter (  ) is assumed for the micelle model, which is described by a Gaussian distribution.Thus, the polydispersity function in Equation S1 can be represented as: where   is the standard deviation for   .In accordance with Equation S2, the number density per unit volume for the micelle model is expressed as: where  is the total volume fraction of copolymer in the spherical micelles and ( 1 ) is the total volume of copolymer within a spherical micelle [( 1 ) = (  +   )  ( 1 )].

Worm-like micelle model
The worm-like micelle form factor for Equation S1 is given by: where all the parameters are the same as those described in the spherical micelle model (Equation S3), unless stated otherwise.
The self-correlation term for the worm core cross-sectional volume-average radius   is: where and  1 is the first-order Bessel function of the first kind, and a form factor   (,   ,   ) for selfavoiding semi-flexible chains represents the worm-like micelles, where   is the Kuhn length and   is the mean contour length.A complete expression for the chain form factor can be found elsewhere. 6e mean aggregation number of the worm-like micelle,  w , is given by: where   is the volume fraction of solvent within the worm-like micelle cores, which was found to be zero in all cases.The possible presence of semi-spherical caps at both ends of each worm is neglected in this form factor.
A polydispersity for one parameter ( w ) is assumed for the micelle model, which is described by a Gaussian distribution.Thus, the polydispersity function in Equation S1 can be represented as: where   w is the standard deviation for  w .In accordance with Equation S2, the number density per unit volume for the worm-like micelle model is expressed as: where  is the total volume fraction of copolymer in the worm-like micelles and ( 1 ) is the total volume of copolymer in a worm-like micelle [( 1 ) = ( s +  c ) w ( 1 )].

Vesicle model
The vesicle form factor in Equation S1 is expressed as: where all the parameters are the same as in the spherical micelle model (see Equation S3) unless stated otherwise.
The amplitude of the membrane self-term is: where   =   − .It should be noted that Equation S16 differs subtly from the original work in which it was first described. 7The exponent term in Equation S17 represents a sigmoidal interface between the blocks, with a width   accounting for a decaying scattering length density at the membrane surface.The value of   was fixed at 2.5 during fitting.The mean vesicle aggregation number,   , is given by: where   is the volume fraction of solvent within the vesicle membrane, which was found to be zero in all cases.Assuming that there is no penetration of the solvophilic coronal blocks into the solvophobic membrane, the amplitude of the vesicle corona self-term is expressed as: where the term outside the square brackets is the factor amplitude of the corona block polymer chain such that: For the vesicle model, it was assumed that two parameters are polydisperse: the radius from the centre of the vesicles to the centre of the membrane and the membrane thickness (denoted   and   , respectively).Each parameter is considered to have a Gaussian distribution of values, so the polydispersity function in Equation S1 can be expressed in each case as: where   and   are the standard deviations for   and   , respectively.Following Equation S2, the number density per unit volume for the vesicle model is expressed as: where  is the total volume fraction of copolymer in the vesicles and ( 1 ,  2 ) is the total volume of copolymers in a vesicle [( 1 ,  2 ) = (  +   )  ( 1 ,  2 )].

Gaussian chain model
Generally, the scattering cross-section per unit sample volume for an individual Gaussian polymer chain can be expressed as: where  mol is the total molecular volume and ∆ is the excess scattering length density of the copolymer [∆ =  PNAM−PCAm −  water = 1.18 x 10 -10 cm -2 ], where the scattering length density of the copolymer is calculated as . The generalised form factor for a Gaussian polymer chain is given by: where the lower incomplete gamma function is (, ) = ∫  −1 exp(−)   0 and  is the modified variable: Here,  is the extended volume parameter and  g cop is the radius of gyration of the copolymer chain.

1 2𝑇 2 𝑇
is the inner radius of the membrane,   =   + 1  is the outer radius of the membrane ( m is the radius from the centre of the vesicle to the centre of the membrane),